Advances in Botanical Research, Volume 29

Advances in BOTANICAL RESEARCH incorporating Advances in Plant Pathology VOLUME 29 Advances in BOTANICAL RESEARCH ...

Author: J. A. Callow

89 downloads 854 Views 12MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!

Report copyright / DMCA form

DOWNLOAD PDF

Advances in

BOTANICAL RESEARCH incorporating Advances in Plant Pathology

VOLUME 29

Advances in

BOTANICAL RESEARCH incorporating Advances in Plant Pathology Editor-in-Chief J. A. CALLOW

School of Biological Sciences, University of Birmingham, UK

Editorial Board J. H. ANDREWS

University of Wisconsin-Madison, Madison, USA J. S. HESLOP-HARRISON John Innes Centre, Norwich, UK Universite' de Puris-Sud, Orsay, France M. KREIS R. M. LEECH University of York, York, UK R. A. LEIGH University of Cambridge, Cambridge, UK University of California, Riverside, USA E. LORD I. C . TOMMERUP CSIRO, Perth, Australia

Advances in

BOTANICAL RESEARCH incorporating Advances in Plant Pathology Series editor

J. A. CALLOW School of Biological Sciences, University of Birmingham, Birmingham, UK

VOLUME 29

1999

ACADEMIC PRESS San Diego London Boston Ncw York Sydney Tokyo Toronto

This book is printed on acid-free paper Academic Press 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http:/lwww.apnet.com Academic Press Limited 24-28 Oval Road, London NW1 7DX, UK http:llwww.hbuk.co.uWap/ Copyright 0 1999 by ACADEMIC PRESS All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. A catalogue record for this book is available from the British Library

ISBN 0-12-005929-0

Typeset by Keyset Composition, Colchester, Essex Printed in Great Britain by MPG Books Limited, Bodmin, Cornwall

99 00 01 02 03 04 MP 9 8 7 6 5 4 3 2 1

CONTENTS

CONTRIBUTORS TO VOLUME 29 CONTENTS OF VOLUMES 18-28 PREFACE

.............................................................

ix

...............................................................

xi

.........................................................................................................

xxi

The Calcicole-Calcifuge Problem Revisited J . A. LEE I.

............................................................................................

2

I1. Edaphic Factors ................................................................................................ A . Acid Soils ...... ..................................................................................... B . Calcareous Soils .......................................................................................

2 4 4

111.

Introduction .....

Controlled Environment Experimentation on Individual Edaphic Factors ........ A . Aluminium and Acidity .............................................................................. B . Iron and Manganese Toxicity ................................................................... C . Bicarbonate Toxicity and Iron Deficiency ............ ............................... D . Phosphate ........................................................................................... E. Calcium ........................................................... .... F. Nitrogen .......................................................... .... G . Microelements ..........................................................................................

IV. Conclusion ...................................................................................

4 4 10 11

13 14 17 20 22

Acknowledgements ...........................................................................................

25

References ........................................................................................................

25

Ozone Impacts on Agriculture: an Issue of Global Concern M . R . ASHMORE and F. M . MARSHALL I . Introduction

......................................................................................................

I1. Ozone Impacts on Agricultural Crops .............................................................. A . Exposure-Response Studies ..................................................................... 111.

Rural Ozone Levels in Developing Countries ..................................................

32 33 34 36

vi

CONTENTS

IV. Direct Evidence of Adverse Effects on Crops ................................................. A . Studies with Field Chambers .................................................................... B . Studies with Ozone Protectant Chemicals ................................................

39 39 41

V. Responses to Ozone of Tropical Crops and Cultivars ........................................ A. Experimental Studies .................................................................................. B . Factors Influencing Ozone Sensitivity in the Field ....................................

43 43 45

VI . Future Concentrations and Impacts of Ozone ....................................................

46

VII . Conclusions ........................................................................................................

46

Acknowledgements ............................................................................................

48

References ..........................................................................................................

49

Signal Transduction Networks and the Integration of Responses to Environmental Stimuli G . I . JENKINS I . Introduction ........................................................................................................ A . Networks Versus Pathways ......................................................................... B . Achieving an 'Appropriate' Response ........................................................ 11.

.

111

54 55 56

Interactions Within Signalling Networks ............................................................ A. Evidence of Negative Regulation ............................................................... B . Evidence for Synergism ..............................................................................

57 57 63

Approaches to Identify the Mechanisms Involved in Interactions Between Signalling Pathways ..........................................................................................

67

Conclusions ........................................................................................................

69

Acknowledgements ............................................................................................

70

References

..........................................................................................................

70

Mechanisms of Na' Uptake by Plants A. AMTMANN and D. SANDERS 1. Introduction ........................................................................................................ A. Salinity Toxicity and Salinity Tolerance ..................................................... B . Exclusion . Uptake and Sequestration of Na+ .............................................

76 76 77

I1. Electrochemical Potential Differences for Na+ Across the Plasma and Vacuolar Membranes ......................................................................................................... 78

CONTENTS

vii

111. Carrier-mediated Entry of Na’ ...........................................................................

80

IV. Channel-mediated Entry of NaC ......................................................................... A. Ionic Selectivity of Ion Channels B. Inward-Rectifying Channels .... C. Outward-Rectifying Channels . D. Voltage-Independent Channels E. Co-residency of Different Channel Types ..................................................

82

89

V. Contributions of Channel Types to Na+ Entry in Physiological Conditions ...... 91 9I A. Semiquantitative Dissection of Fluxes ....................................................... B. Relative Activity of Different Channel Types Determines Rate of Na+ Uptake ......................................................................................................... 95 VI. Regulation of Monovalent Cation Influx Across the Plasma Membrane ........... 96 ....................................................................... 97 A. Voltage ......................... B. External Ca” and pH .. C. Cytosolic Ca’+ and pH ........ D. External and Cytosolic Na+ E. ATP .............................. F. Other Regulators .......... VII. Comparison of Salt-Sensitive and Salt-Tolerant Genotypes or Cell Lines ....... 103 VIII. Future Work ......................................................................................................

103

Acknowledgements ...........................................................................................

104

References ........................................................................................................

104

The NaC1-induced Inhibition of Shoot Growth: The Case for Disturbed Nutrition with Special Consideration of Calcium Nutrition D. B. LAZOF and N. BERNSTEIN I.

11.

Ill.

IV.

Introduction to the Inhibition of Shoot Growth by Salinity ............................. A. Growth Inhibitions: General Considerations ............................................. B. NaCI-induced Inhibition of Shoot Growth: General Hypotheses ............. C. A Nutritional Effect of NaCl on Shoot Growth ........................................

115

115 116

119

Inhibition of Shoot Growth in Dicots and Monocots ...................................... A. The Timing of the Growth Inhibition .... B. Salinity Effects on Cell Extension ......... C. Salinity Effects on Primordium Formatio D. Salinity Effects on Cell Division in Leaves

12 I

NaCI-induced Disruptions of Nutrient Transport ............................................. A. Influence of Some Experimental Conditions ..... . B. Effects on Whole Shoot Nutrient Accumulation ......................................

I26 126 128

Nutrient Transport to Growing Shoot Tissue Under Salinity ...........................

132

...

CONTENTS

Vlll

A. B. C. D. E. F. G. H.

Protection of Growing Tissues ................................................................. Levels of Na and K in Young Tissues ...................................................... Disturbed Ca Status in Young Tissues ...................................................... Other Nutrient Disruptions in Young Shoot Tissues ................................ Effects in Young Tissues Compared to Effects in Mature Tissues ........... Genotypic Salinity Effects in Young Tissues ........................................... Lactucu surivu: a Model Dicot System .................................................... Summary: Salinized Nutrition of Growing Shoot Tissues .......................

V. The Shoot Meristems: Special Nutrient Transport Challenges ......................... A. The Nutrition of Rapidly Dividing Cells: Possible Effects of Salinity B . Transport to Zones Proximal to the Meristem in Poaceae .......................

....

146 148

149

VI . Phloem Transport and Ion Recirculation Under Salinity ................................. A. Remobilization of Nutrients from Ageing Shoot Tissues. ‘Long-term Recirculation’ ............................................................................................ B. XylemRhloem Transfer, ‘Short-term Recirculation’ ................................ C. Calcium Recirculation in the Shoot .......................................................... D. Summary of Salinization and Recirculation ............................................. VII .

133 133 137 138 139 141 141 143

Salinization and Shoot Nutrition: Specific Nutrients ........................................ A. Potassium ............... ........ B. Calcium .................. ........ C. Magnesium ............................................................................................... D. Phosphorus ................................................................................................ E . Nitrogen ............................... ............................................................. F. Micronutrients ...........................................................................................

150 151 154 155 156 157 157 157 158 158 160 162

VIII. The Study of Nutrient Status and Transport on the Microscale ........ A. Kinematic Growth Analysis and Elemental Deposition Rates ................ B . Microdissection ......................................................................................... C . Specimen Preparation Considerations ...................................................... D. Electron Probe X-ray Microanalysis ......................................................... E . Secondary Ion Mass Spectrometry ........................... ........ ... F. Some Other Microanalytical Techniques ..................................................

162 163 166 166 167 168 170

IIIX . Summary and Future Prospects ........................................................................ A . Reassessment of Current Status ................................................................ B . Model Systems .. C. In Situ Elemental ...........................................

171 171 173 174

Acknowledgements References

....

.......

..... 175

........................................................................................................

175

AUTHOR INDEX ............................................................................................

191

............................................................................................

203

SUBJECT INDEX

Plates are located between p p . 74-75 .

CONTRIBUTORS TO VOLUME 29

A. A M T M A " The Plant Laboratory, Biology Department, PO Box 373, University of York, York YO1 SYH? UK M. R. ASHMORE Department of Environmental Science, University of Bradford, West Yorkshire, BD7 IDP, U K N. BERNSTEIN Institute of Soil Water; The Volcani Center; PO Box 6, Bet Dagan 50250, Israel G. I. JENKINS Plant Molecular Science Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Bower Building, University of Glasgow, Glasgow G I 2 8QQ, UK D. B. LAZOF Department of Chemistry, CB 3290, University of North Carolina, Chapel Hill, North Carolina 27599, USA J. A. LEE Department of Animal and Plant Science, University of Shefield, Shefield SIO ZTN, UK F. M. MARSHALL Centre for Environmental Technology, Imperial College of Science, Technology and Medicine, 48 Princes Gardens. London SW7 2PE, UK D. SANDERS The Plant Luboratoq Biology Department, PO Box 373, University of York, York YO1 5YW UK

This Page Intentionally Left Blank

CONTENTS OF VOLUMES 18-28

Contents of Volume 18 Photosynthesis and Stornatal Responses to Polluted Air, and the Use of Physiological and Bacterial Responses for Early Detection and Diagnostic Tools

H. SAXE Transport and Metabolism of Carbon and Nitrogen in Legume Nodules J. G. STREETER Plants and Wind

P. VAN GARDINGEN and J. GRACE Fibre Optic Microprobes and Measurement of the Light Microenvironment within Plant Tissues T. C. VOGELMANN, G. MARTIN, G. CHEN and D. BUTTRY

Contents of Volume 19 Oligosaccharins

S. ALDINGTON and S. C. FRY Are Plant Hormones Involved in Root to Shoot Communication?

M. B. JACKSON Second-Hand Chloroplasts: Evolution of Cryptomonad Algae G. I. McFADDEN

The Gametophyte-Sporophyte Junction in Land Plants R. LIGRONE, J. G. DUCKETT and K. S. RENZAGLIA

xii

CONTENTS OF VOLUMES 18-28

Contents of Volume 20 Global Photosynthesis and Stomatal Conductance: Modelling the Controls by Soil and Climate E I. WOODWARD and T. M. SMITH

In vivo NMR Studies of Higher Plants and Algae

R. G. RATCLIFFE Vegetative and Gametic Development in the Green Alga Chlamydomonas H. VAN DEN ENDE

Salicylic Acid and its Derivatives in Plants: Medicines, Metabolites and Messenger Molecules W. S. PIERPOINT

Contents of Volume 21 Defence Responses of Plants to Pathogens E. KOMBRINK and I. E. SOMSSICH

On the Nature and Genetic Basis for Resistance and Tolerance to Fungal Wilt Diseases of Plants C. H. BECKMAN and E. M. ROBERTS

Implication of Population Pressure on Agriculture and Ecosystems A. H. EHRLICH

Plant Virus Infection: Another Point of View G. A. DE ZOETEN

The Pathogens and Pests of Chestnuts S. L. ANAGNOSTAKIS

CONTENTS OF VOLUMES 18-28

Fungal Avirulence Genes and Plant Resistance Genes: Unraveling the Molecular Basis of Gene-for-Gene Interactions P. J. G. M. DE WIT

Phytoplasmas: Can Phylogeny Provide the Means to Understand Pathogenicity? B. C. KIRKPATRICK and C. D. SMART

Use of Categorical Information and Correspondence Analysis in Plant Disease Epidemiology S. SAVARY, L. V. MADDEN, J. C. ZADOKS and H. W. KLEIN-GEBBINCK

Contents of Volume 22 Mutualism and Parasitism: Diversity in Function and Structure in the “Arbuscular” (VA) Mycorrhizal Symbiosis F. A. SMITH and S. E. SMITH

Calcium Ions as Intracellular Second Messengers in Higher Plant A. A. R. WEBB, M. R. McAINSH, J. E. TAYLOR and I. M. HETHERINGTON

The Effects of Ultraviolet-B Radiation on Plants: A Molecular Perspective B. R. JORDAN

Rapid, Long-Distance Signal Transmission in Higher Plants M. MALONE

Keeping in Touch: Responses of the Whole Plant to Deficits in Water and Nitrogen Supply A. J. S. McDONALD and W. J. DAVIES

...

Xlll

xiv

CONTENTS OF VOLUMES 18-28

Contents of Volume 23 PATHOGEN INDEXING TECHNOLOGIES The Value of Indexing for Disease Control Strategies D. E. STEAD, D. L. EBBELS and A. W. PEMBERTON

Detecting Latent Bacterial Infections S. H. DE BOER, D. A. CUPPELS and R. GITAITIS

Sensitivity of Indexing Procedures for Viruses and Viroids H. HUTTINGA

Detecting Propagules of Plant Pathogenic Fungi S. A. MILLER

Assessing Plant-Nematode Infestations and Infections K. R. BARKER and E. L. DAVIS

Potential of Pathogen Detection Technology for Management of Diseases in Glasshouse Ornamental Crops

I. G. DINESEN and A. VAN ZAAYEN Indexing Seeds for Pathogens J. LANGERAK, R. W. VAN DEN BULK and A. A. J. M. FRANKEN A Role for Pathogen Indexing Procedures in Potato Certification S. H. DE BOER, S. A. SLACK, G. VAN DEN BOVENKAMP and I. MASTENBROEK

A Decision Modelling Approach for Quantifying Risk in Pathogen Indexing C. A. LEVESQUE and D. M. EAVES

Quality Control and Cost Effectiveness of Indexing Procedures C. SUTULAR

CONTENTS OF VOLUMES 18-28

Contents of Volume 24 Contributions of Population Genetics to Plant Disease Epidemiology and Management M. G. MILGROOM and W. E. FRY

A Molecular View through the Looking Glass: the Pyrenopeziza brussicae-Brussica Interaction A. M. ASHBY The Balance and Interplay Between Asexual and Sexual Reproduction in Fungi M. CHAMBERLAIN and D. S. INGRAM The Role of Leucine-Rich Repeat Proteins in Plant Defences D. A. JONES and J. D. G. JONES Fungal Life-styles and Ecosystem Dynamics: Biological Aspects of Plant Pathogens, Plant Endophytes and Saprophytes R. J. RODRIGUEZ and R. S. REDMAN

Cellular Interactions between Plants and Biotrophic Fungal Parasites M. C. HEATH and D. SKALAMERA Symbiology of Mouse-Ear Cress (Arubidopsis thulianu) and Oomycetes E. B. HOLUB and J. L. BEYNON

Use of Monoclonal Antibodies to Detect, Quantify and Visualize Fungi in Soils F. M. DEWEY, C. R. THORNTON and C. A. GILLIGAN Function of Fungal Haustoria in Epiphytic and Endophytic Infections

P. T. N. SPENCER-PHILLIPS

xv

xvi

CONTENTS OF VOLUMES 18-28

Towards an Understanding of the Population Genetics of Plant-Colonizing Bacteria B. HAUBOLD and P. B. RAINEY

Asexual Sporulation in the Oomycetes A. R. HARDHAM and G. J. HYDE

Horizontal Gene Transfer in the Rhizosphere: a Curiosity or a Driving Force in Evolution? J. WOSTEMEYER, A. WOSTEMEYER and K. VOIGT

The Origins of Phytophthoru Species Attacking Legumes in Australia J. A. G. IRWIN, A. R. CRAWFORD and A. DRENTH

Contents of Volume 25 THE PLANT VACUOLE The Biogenesis of Vacuoles: Insights from Microscopy F. MARTY

Molecular Aspects of Vacuole Biogenesis D. C. BASSHAM and N. V. RAIKHEL

The Vacuole: a Cost-Benefit Analysis J. A. RAVEN

The Vacuole and Cell Senescence P. MATILE

Protein Bodies: Storage Vacuoles in Seeds G. GALILI and E. M. HERMAN

Compartmentation of Secondary Metabolites and Xenobiotics in Plant Vacuoles M. WINK

CONTENTS OF VOLUMES 18-28

xvii

Solute Composition of Vacuoles R. A. LEIGH

The Vacuole and Carbohydrate Metabolism C. J. POLLOCK and A. KINGSTON-SMITH

Vacuolar Ion Channels of Higher Plants G. J. ALLEN and D. SAUNDERS

The Physiology, Biochemistry and Molecular Biology of the Plant Vacuolar ATPase U. LUlTGE and R. RATAJCZAK

The Molecular and Biochemical Basis of Pyrophosphate-Energized Proton Translocation at the Vacuolar Membrane R.-G. ZHEN. E. J. KIM and P. A. REA

The Bioenergetics of Vacuolar H+ Pumps J. M. DAVIES Transport of Organic Molecules Across the Tonoplast E. MARTINOIA and R. RATAJCZAK

Secondary Inorganic Ion Transport at the Tonoplast E. BLUMWALD and A. GELLI

Aquaporins and Water Transport Across the Tonoplast M. J. CHRISPEELS. M. J. DANIELS and A. W E E

Contents of Volume 26 Developments in the Biological Control of Soil-borne Plant Pathogens J. M. WHIPPS

xviii

CONTENTS OF VOLUMES 18-28

Plant Proteins that Confer Resistance to Pests and Pathogens P. R. SHEWRY and J. A. LUCAS

The Net Primary Productivity and Water Use of Forests in the Geological Past D. J. BEERLING

Molecular Control of Flower Development in Petunia hybrids L. COLOMBO, A. VAN TUNEN, H. J. M. DONS and G. C. ANGENENT

The Regulation of C4Photosynthesis R. C. LEEGOOD

Heterogeneity in Stomata1 Characteristics J. D. B. WEYERS and T. LAWSON

Contents of Volume 27 CLASSIC PAPERS The Structure and Biosynthesis of Legume Seed Storage Proteins: A Biological Solution to the Storage of Nitrogen in Seeds D. BOULTER and R. R. D. CROY

Inorganic Carbon Acquisition by Marine Autotrophs J. A. RAVEN

The Cyanotoxins W. W. CARMICHAEL

Molecular Aspects of Light-harvesting Processes in Algae T. LARKUM and C. J. HOWE

Plant Transposable Elements

R. KUNZE, H. SAEDLER and W.-E. LONNIG

CONTENTS OF VOLUMES 18-28

xix

Contents of Volume 28 Protein Gradients and Plant Growth: Role of the Plasma Membrane H+-ATPase

M. G. PALMGREN The Plant Invertases: Physiology, Biochemistry and Molecular Biology Z. TYMOWSKA-LALANNE and M. JSREIS

Dynamic Pleomorphic Vacuole Systems: Are They Endosomes and Transport Compartments in Fungal Hyphae? A. E. ASHFORD

Signals in Leaf Development T. P. BRUTNELL and J. A. LANGDALE

Genetic and Molecular Analysis of Angiosperm Flower Development V. F. IRISH and E. M. KRAMER

Gametes, Fertilization and Early Embryogenesis in Flowering Plants C. DUMAS, F. BERGER, J. E.-FAURE and E. MATTHYS-ROCHON

This Page Intentionally Left Blank

PREFACE

One of the classical problems of experimental plant ecology for much of this century, has been the elucidation of mechanisms which control the extreme growth preferences many plants show for either acidic or calcareous soils. The chapter by Lee evaluates both the historical evidence on this ‘calcicole-calcifugeproblem’, and more recent information on plant ion transport processes and calcium signalling. The potential contribution of molecular technologies is considered. The article stresses the importance of studying the interactions between edaphic factors rather than single factors and emphasises the important, but rather neglected contribution of the rhizosphere microflora. The impact of ozone pollution on crop production in the developed world is well documented but its importance in the more vulnerable economies of the developing countries of Asia, Africa and Latin America is less clear. The article by Ashmore and Marshall considers the effects of ozone on the physiology and yield of crop plants and identifies thresholds for impacts on crops of less-developed countries. The article then shows that actual ozone concentrations in LDCs may indeed reach inhibitory concentrations for sensitive species, that yields of staple crops may be adversely affected, and that there is considerable potential for a worsening situation over the next two decades. Ozone impacts on agriculture must now be considered as a global issue and the authors identify a clear and urgent need for collaborative international experimental programmes. As sedentary organisms the survival of higher plants depends critically on their abilities to detect and respond appropriately to a range of environmental signals, including those which are potentially stressful such as UV light and molecules produced by pathogens. Such responses involve mechanisms which detect the primary signals and which transduce those signals into a form that can promote the coordinated expression of specific sub-sets of genes. The article by Jenkins shows that these signals are not perceived and transduced in isolation. Rather, there are mechanisms for interaction within signal transduction networks which allow the plant to exhibit a response which is coordinated and integrated with other signals and processes including those which regulate normal development. A strength of the article is an evaluation of the exciting information emerging from the combined use of newer cell technologies such as microinjection, and the measurement of ion channel activities and intracellular calcium transients, with the approaches of molecular genetics including transgenic manipulation of the signalling pathway. One of the major limitations to crop growth on marginal soils, and some agriculture-intensive, irrigated soils is salinity. Lack of understanding of the mechanisms of salt tolerance and salt-induced inhibition of growth have hampered attempts to develop new crop cultivars with improved salt tolerance, and two articles

xxii

PREFACE

consider these two complementary aspects of the problem. The article by Amtmann and Sanders explores salinity tolerance from the perspective of recent advances in our knowledge of the sodium transport pathways across cell membranes. Data on selectivity, conductance, abundance and regulation of major cation uptake channel types is considered and integrated into a simple model which can be used to explore physiological questions on how some plants cope with saline environments. The article by Lazof and Bernstein focuses on the inhibition of shoot growth by NaCl which is one of the earliest responses to salinity. The physiological basis of this is unclear and the main thesis of the authors is that physiologists have tended to concentrate on whole or mature tissues, or tissues of ill-defined developmental and physiological status and that insufficient attention has been paid to the events in dividing and rapidly expanding cells and tissues. They hypothesise that the primary cause of salt-induced growth inhibition is a disturbance of mineral nutrition in these minute zones. Recommendations are made for advanced analytical methods that might be used to quantify alterations in nutrient transport and status within areas of rapid growth. As usual the Editor would like to thank all the contributors to this volume, for their patience and cooperation in making his task easier. J. A. Callow

The Calcicole-Calcifuge Problem Revisited

J. A. LEE

Department of Animal and Plant Sciences, University of Shefield S10 2TN, UK

I.

Introduction ...........................................................................................................

2

...............................

2 4 4

11. Edaphic Factors

A. B.

Acidic Soils .................................................................................................... Calcareous Soils ............

111. Controlled Environment Experimentation on Individual Edaphic factors ............. 4 4 A. Aluminium and Acidity ....................... B. Iron and Manganese Toxicity ...................................................................... 10 C. Bicarbonate Toxicity and Iron Deficiency 11 D. Phosphate ................................................... 13 E. Calcium . .............................. 14 F. Nitrogen ............................. 17 G. Microelements .............................................................................................. 20

IV. Conclusion ........................................................................................................... Acknowledgements .............................................................................................. References .................................

22 25 25

The adaptations shown by plants to growth in acidic and calcareous soils have ,fascinated ecologists during much of the 20th Century, but have not so f a r been elucidated entirely. This paper describes the major edaphic factors operating in these soils, arid discusses recent advances in our understunding of the udaptutions of calcicole arid calcifuge species to them. It concentrates on an evaluation of the results from controlled environment experimentation on variations in individual edaphic juctors. Adaptations to the ,following edap1zic.factor.sare considered in detail: low p H , toxicities of aluminium, bicarbonate and manganese deficiencies and to-ricities qf calcium and iron, and nitrogen and phosphorus availability. Although most experiments have coricentruted on udaptutions to individual eduphic factors, the importance of studying interactions between these factors is stressed. The paper also eniphasizes the potential importance of rhizosphere microorganisms in modibing plant Advances In Botanical Research Vul 29 incorporating Advances In Plant Pathology ISBN 0- 12-005929-0

Copyright 0 199’) Academic Press 411right,

reproduction

any rOml reserved

2

J. A. LEE

responses to edaphic factors. Important recent advances in our understanding of plant adaptations to aluminium and calcium supply in particular highlight the need to harness molecular biological tools and modern methods of studying ion transport processes to improve our understanding of the major factors involved in determining the distribution of plants on acidic and calcareous soils.

I. INTRODUCTION The origin of the modern era of experimental plant ecology can be traced to Tansley (1917) and was based on attempts to understand the factors governing plant distribution at a small scale. Tansley observed that Galium saxutile L. was confined to deep brown Podzolic soils on the limestone peneplain of Derbyshire, UK whereas Gulium stemeri Ehrend was confined to shallow rendzinas on the limestone dales. The deep acidic soils of the peneplain were later demonstrated to result from the deposition of wind-blown loess at the time of the last glaciation (Pigott, 1962). Although these two soil types are frequently found only a few metres apart, their floras are always distinct. Tansley conducted an experiment in the Cambridge Botanic Garden sowing the seed of the two Galium species alone and in competition with one another on a range of soils. Gulium saxutile grew much more vigorously on acidic soils when grown in monocultures, but a few survived and flowered on the calcareous soil. Gulium stemeri grew well on the calcareous soil, but some seedlings survived even in competition with Galium saxatile on acid peat. However, when grown in competition with Galium stemeri, Gulium saxatile seedlings only survived on acidic soils. Tansley concluded that soil factors combined with competition resulted in the distribution of the two species in the field. It was not until the 1950s that field experiments were first established to test the validity of Tansley’s findings. Rorison (1960a), in an experiment on the chalk and greensand of southern England, demonstrated that competition from other species was not the primary factor affecting seedling survival of calcicoles and calcifuges in respectively acid and calcareous grassland. Later experiments by Grime and his co-workers (see e.g. Grime and Curtis, 1976) demonstrated, for example, that seeds of calcifuge species germinated on calcareous soils in Derbyshire, but seedling mortality was particularly associated with frost and drought. The field experiments of Grime and Rorison supported the earlier view of Hope-Simpson (1938) that the primary factor governing the distribution of calcicoles and calcifuges was soil chemistry.

11. EDAPHIC FACTORS The fact that large differences in soil chemistry can occur over differences of only a few metres in the limestone dales of Derbyshire is illustrated in Table. I. The extractable calcium is c.24 times, the magnesium c.6 and the ammonium c.2 times greater in the rendzina than in the brown podzolic soil. The latter has no detectable

TABLE I Some extractable element contents ( p g g-’ soil) from two soils over limestone at Coombesdale, Derbyshire, UK. Cations and phosphorus were extracted with 1M ammonium acetate (pH 7.0), and nitrate-N and ammonium-N with IM KCI. Figures are means of a minimum of 10 samples (cations and phosphorus) and 8 samples (N03-N and NH3-N)2 1 S.D. pH was determined in a 1:1 (wt) aqueous suspension. Data of J. R. L e a h

Rendzina Podzolic brown earth

Ca

Mg

Fe

K

P

NOyN

N€&-N

PH

6958 ? 472 291 5 2 9 1

172 2 13 2925

1?1 25 5 2

8529 95 ? 35

3.8 ? 1.6 6.0 5 4.6

1922 0

21 ? 3 921

7.0 5 0.1 4.65 0.2

4

J. A. LEE

nitrate and a 25 times greater extractable iron content than the former. In contrast the two soils have similar extractable potassium and phosphorus contents. However, much of our knowledge of the chemical composition of soil solutions and its effects on plant growth in acidic and calcareous soils comes from agriculture (see e.g. Hewitt, 1952). This information can be summarized as follows. A. ACIDIC SOILS

Soil acidity factors include deficiencies of calcium, magnesium, potassium and molybdenum, increased solubility and toxicity of aluminium, manganese and iron, reduced availability of phosphate, and an impaired nitrogen cycle. In agricultural soils (other than those on periodically waterlogged acid sulphate soils) there is no evidence that hydrogen ion concentration per se is directly damaging to plant growth. B. CALCAREOUS SOILS

Shallow soils over chalk or limestone are typically highly porous, freely draining and saturated with calcium carbonate. The predominant ions in the soil solution are Ca2* and HCO;, the concentration of the latter in particular being dependent on the partial pressure of carbon dioxide in the soil atmosphere. Associated with a pH of the soil solution close to neutrality (pH 7-8), low plant availability of iron, cobalt, boron and phosphate are potentially important factors. The predominance of Ca” in the soil solution may also pose problems for the uptake of, for example, K + . However, in calcareous soils it is assumed that there is no impairment of the nitrogen cycle, and that NO3- is the major form of available nitrogen for plants (but see Table I) whereas NH: predominates in acidic soils.

111. CONTROLLED ENVIRONMENT EXPERIMENTATION ON INDIVIDUAL EDAPHIC FACTORS The 1960s represented the most intensive period of experimentation on the calcicolecalcifuge problem. This reflected the new availability of improved controlled environment facilities and the great interest in autecological problems. Emphasis was very much placed on an understanding of the relative importance of individual soil factors in determining plant distribution. A strong feature of these investigations was a comparison of the responses of calcicole and calcifuge species to individual edaphic factors. A. ALUMINIUM AND ACIDITY

The potential importance of aluminium as a differential edaphic factor can be judged by the fact that it is the most abundant metallic element in the soil, it is toxic to many plants at low concentrations in solution, and is present mostly in insoluble

THE CALCICOLE-CALCIFUGE PROBLEM REVISITED

5

forms above pH 5.0. Rorison (1960b) demonstrated that the calcicole species Scahiosa columburia L. produced stunted roots when grown on an acidic sand which were similar to those produced when Scabiosu was grown on a solution containing 5 0 m g l - ' Al. He suggested that the principal cause of the failure of Scnbiosa on acidic soils was aluminium toxicity. Subsequently a series of investigations demonstrated that calcicole species showed marked growth inhibition by aluminium in solution, whereas calcifuge species were largely unaffected or even stimulated at low concentrations (see e.g. Clarkson, 1966). The importance of aluminium as a soil acidity factor can also be inferred from intraspecific studies. Thus Davies and Snaydon ( 1973) demonstrated that populations of Anthoxanthurn odoraturn L. in acidic and calcareous (limed) plots from the Park Grass Experiment at Rothamsted differed in their response to aluminium in solution. Acidic populations showed little or no inhibition of root growth by up to 54 mg I F ' Al whereas calcareous populations were inhibited throughout the range of aluminium concentrations used. This difference in response to aluminium evolved within 65 years of liming treatments being imposed, pointing to the importance of aluminium as a soil acidity factor. New information on the toxicity of aluminium species in solution has led to an awareness of the toxicity of A17+,the extreme toxicity of the Al13polymer and the non-toxicity of AI(0H)'- and various aluminium chelates (see e.g. Kinraide and Parker, 1990; Kinraide, 1991; Kinraide and Ryan, 1991). The major factors affecting the rate of dissociation of minerals in soils appear to be pH and the availability of ions to react with the dissolving surface. The release of aluminium from kaolinite, for example, is a function of pH in the range where H+ ions are adsorbed by the clay, and reaches a maximum at pH <3.5 where no more H+ ions can be absorbed (Wieland and Stumm, 1992). However, predicting, aluminium species solubility from mineral dissolution and precipitation remains difficult (Ritchie, 1994). No single form of solid aluminium controls aluminium in solution, and the pathway of aluminium before it reaches the soil solution may involve retention and release from a number of different sinks. The maintenance of aluminium in solution depends in the short term (days) on exchangeable aluminium which is in turn replenished by interlayer and organically bound aluminium, and is ultimately dependent on mineral dissolution. Thus the chemistry of aluminium in soils is complex, but the extent of phytotoxicity is usually related to the activity of monomeric inorganic aluminium species in solution (see e.g. Cameron ef al., 1986). However, using NMR techniques the presence of AII3 has been reported in an acidic forest soil (Hunter and Ross, 1991), and further research is necessary to assess its ecological importance. Most experiments have examined the sensitivity of plants to A17+, and rapid responses (minutes) have concentrated on effects on plasma membrane transport processes. Among the effects that have been observed in root apices are the blocking of Ca" permeable channels (Pineros and Tester, 1993) resulting in prolonged reduction in Ca" concentration in the cytosol (Fig. 1 ) (Jones et d.,1998), blocking K' channels (Gassmann and Schroeder, 1994), interference with the phosphoinositide signalling pathway through inhibition of

6

J. A. LEE

200

+ (v

h

([1

0

V

-

.or - c

150

0 -

v, 0 c

100

h

0

50

0

Time (min) Fig. 1. Effect of A1 on [Ca2+Ic,,in BY-2 tobacco cells. Cells were acid loaded with Indo-1 and maintained in a perfusion chamber on a microscope stage. Cells were perfused with 0, 50, 100 and 200 pM AICI3 and Ca2+ distribution determined by confocal ratio imaging. After 20 min of A1 treatment the cells were perfused with Al-free medium and the effect on [Ca2'] cyt was monitored. Results are means 2 SE, nr30. From Jones ef al. (1998).

phospholipase C activity (Jones and Kochian, 1995), and inhibition of nitrate uptake and increase in net Hf release (Calba and Jaillard, 1997) (Fig. 2). Organically complexed aluminium species are less toxic than inorganic ones, and thus organic matter may ameliorate aluminium phytotoxicity (see e.g. Bartlett and Riego, 1972). Despite the recognition of the importance of aluminium toxicity as a soil acidity factor over many years, there is still no consensus as to the tolerance mechanisms employed by crop or calcifuge species (see e.g. Kochian, 1995 for a review). Tolerance mechanisms proposed during the 1960s, for example the importance of aluminium binding mechanisms in the roots of calcifuges (Grime and Hodgson, 1969) remain largely untested, and this at least in part reflects the complex chemistry of aluminium and the difficulties this imposes for experimentation. Some authors concerned with crop species (e.g. Taylor, 1995) have attempted to distinguish between external exclusion and internal tolerance mechanisms on the site of metal detoxification or immobilization. Such distinctions can be difficult to draw because several processes which might result in the exclusion of aluminium or in its detoxification have their origin within the plant. External mechanisms include the unusually high production of root cap mucilage (Johnson and Bennet, 1991; Puthota et al., 1991), cell wall binding, the selective permeability of the plasma membrane, exudation of chelator ligands and the formation of a plant-

7

THE CALCICOLE-CALCIFUGE PROBLEM REVISITED

600

--

500

T1

7

-ul

5

400

Y

X

-3

LL

300

200 0

10

20

30

40

50

AI content in root (prnolg-1)

and H+ release (m) by an AI-tolerant cv of maize (C525M)vs. Fig. 2. NO; uptake (0) A1 content in root. The relationship between Ht release and NO; uptake is: JH+ = -1.006 JNOl + 878.6 (? = 0.927). From Calba and Jaillard (1997).

induced pH barrier in the rhizosphere. This is particularly true of chelator ligands since chelation within the cytoplasm is one of several proposed internal tolerance mechanisms. Other proposed internal mechanisms include the induction of aluminium-binding compounds similar to phytochelatins (Kochian, 1993, compartmentation in the vacuole, and the evolution of aluminium-tolerant enzymes. However, aluminium-tolerant enzymes need not be confined to the cytosol. Woolhouse (1969) showed that acid phosphatase activity in cell wall preparations of edaphic ecotypes of Agrostis cupillaris L. was inhibited by aluminium in the range 0-100 pM,but at low concentration in particular (up to 25 pM),phosphatase activity in preparations from the acid soil ecotype was much less inhibited than that from the calcareous soil ecotype. Tolerance in some species at least is likely to result from a combination of exclusion and internal mechanisms, and there is a great need for further studies of these mechanisms in calcifuge species. Much recent work has concentrated on the use of cell cultures to understand internal resistance mechanisms of crop species, but the work has been bedevilled by experimental conditions which favour the formation of insoluble or non-toxic aluminium species, and there have been difficulties in consistently expressing cultivar difference in aluminium tolerance at the cellular level (see e.g. Taylor, 1995 for Phaseolus vulgaris L.). These difficulties have also complicated efforts to use cell cultures to produce aluminium-tolerant cell lines from callus with a view to the regeneration of aluminium-tolerant plants. If nothing else, cell culture experimentation has highlighted the need for experimental designs which provide control of the species of aluminium present.

8

J. A. LEE

Aluminium tolerance has been attributed by some authors to a single dominant gene (see e.g. Reid, 1971) and by others to polygenic control (see e.g. Caradus and Mackay, 1995). Aniol and Gustafson (1984) showed that aluminium tolerance in Chinese spring wheat could be linked to seven different chromosome arms from the A, B and D genomes indicating the potential genetic complexity of tolerance mechanisms. However, there must be a range of different tolerance mechanisms and genetic controls. For example, it would appear unlikely that aluminium hyperaccumulators, e.g. tea Camellia sinensis, employ similar mechanisms to those of calcifuges which largely exclude the element from their shoots, and those species which effectively exclude aluminium from the plant. Most recent attempts to understand aluminium tolerance mechanisms (see e.g. Delhaize and Ryan, 1995 for a review) have concentrated on crop species. Arguably the most interesting of these is a study of near-isogenic lines of Triticurn aestivurn L. (Delhaize et al., 1993a,b). In these studies aluminium-sensitive backcross isolines accumulated more aluminium in root apices than aluminium-resistant isolines. Yermiyahu et al. (1997) also showed plasma membrane vesicles from an Al-sensitive wheat cultivar absorbed more aluminium than vesicles from a resistant cultivar. Further, Delhaize and his co-workers showed that roots of the aluminium resistant isoline exuded 5 to 10 times more malate than those of the aluminiumsensitive line in response to addition of aluminium to the nutrient solution (Fig. 3). Aluminium-tolerant and sensitive seedlings had similar root malate concentrations, suggesting that aluminium stimulated the synthesis, and possibly also the exudation, of malate. Malate exudation probably occurs via plasma membrane anion channels (Delhaize and Ryan, 1995). At cytoplasmic pH (c. pH 7.0) malate exists as an anion and is transported down the electrochemical gradient. Activation by aluminium of a malate or citrate anion channel would allow a potentially large passive efflux of the anions. However, the cytoplasmic malate or citrate pool is likely to be small, and a large efflux would require continued replenishment either through metabolism or through the activation of tonoplast anion channels. Ryan er al. (1995) showed that several plant anion-channel antagonists inhibited aluminium-enhanced malate efflux. Papemik and Kochian (1997) also showed that aluminium-induced depolarization of wheat root cap cell membrane potentials is probably linked, but not sufficient to trigger malate release. The involvement of anion channels in malate exudation and the possible gating mechanisms requires further investigation. Delhaize el al. (1993b) demonstrated that malic acid added to nutrient solutions (up to 400 pM) was able to protect aluminium-sensitive seedlings from aluminium toxicity (at 50 pM). A similar observation was made by Bartlett and Riego (1972) who showed that citric acid protected maize roots from aluminium toxicity, and resulted in a much lower uptake of aluminium into the plant. However, a problem in the interpretation of laboratory organic acid exudation studies is to understand the importance of the effect in rhizospheres in soil where organic acids may appreciably affect microbial activity and conversely microbes may appreciably influence the concentrations of a wide range of soluble organic compounds.

THE CALCICOLEXALCIFUGE PROBLEM REVISITED

= O &50 OO

9

Al-Sensitive I

I

100

150

2(

Aluminium added (pMj

Fig. 3. Effect of A1 concentration on excretion of rnalic acid by Al-tolerant ( 0 )and Al-sensitive (0) wheat seedlings. Seedlings were incubated for 24 h in nutrient solution containing various concentrations of Al. Error bars denote the range of the mean from duplicate flasks where the bars exceed the size of the symbol. From Delhaize et al. ( 1993b).

An example of one such interaction is the work of Christiansen-Weniger et al. ( 1992) who showed that an aluminium-tolerant wheat cultivar exuded organic acids

at three times greater rates than an aluminium-sensitive cultivar. Rhizospheres of the former had a higher concentration of malic, oxalic and succinic acids, and a much higher capacity for associative nitrogen fixation, these acids being potential substrates of Azospirillum species. Many workers have assumed that there is no effect of pH per se on the growth of calcifuges. This results from the fact that many freely drained acidic soils have pH values in water of 4.04.5, and the classic investigation of the effects of hydrogen ions on the growth of three agricultural species by Arnon and Johnson (1942) revealed no effects of hydrogen ions on growth between pH 4.0 and 8.0. However, there are many acidic soils with pH values in at least some horizons of 3.54.0. Some of these have been affected by a long history of acidic deposition. Anderson and Brunet (1993) examined the sensitivity to Ht and A13' of the woodland grass Brumopsis benekenii (Lange) Holub. They showed that root growth in flowing cultures decreased within the range pH 4.5-3.5 with a critical threshold at pH 3 . 8 4 0 (Fig. 4). Root growth began to be inhibited at 2 0 p M A l . These workers concluded that both A13 ' and H t are important as limiting factors for the distribution of the grass in southern Sweden. Another Swedish study (FalkengrenGrerup and Tyler, 1993) concluded that high H concentrations of the soil solution in nior humus (pH c. 3.6) precludes the establishment and growth of many understorey species in beech forests. It is probable that ecologists have neglected

J. A. LEE

a

3 5

(a)

38

i

4 0

42

Solution pH

I

leo

r

iL bc

4 5

3 5

0))

3 8

4 0

42

45

Solution pH

Fig. 4. Biomass (dry weight) of (a) new roots, grown during experiments, and (b) shoots of Bromus benekenii (2SD) at different solution pH. Different letters indicate significant differences among treatments (ANOVA, Tukey test, P < 0.05, n = 27). From Anderson and Brunet (1993). the ecological importance of hydrogen ions per se as an edaphic factor in organic surface horizons of acidic soils. B. IRON AND MANGANESE TOXICITY

Evidence that either of these polyvalent cations are important toxicity factors in well-aerated acidic soils is sparse. Mahmoud and Grime (1977), in a study of several grass species, showed that the growth of Deschampsia flexuosa (L.) Trin. was stimulated by concentrations of manganese in water culture in the range (0200 mg 1-' whereas that of the calcicole Arrhenatherum elatius (L.) P. Beauv. ex J.S. and C. Presl was strongly inhibited. Although this may suggest that calcicoles and calcifuges in general differ in tolerance to manganese, reflecting the importance of manganese as a toxicity factor in acidic soils, there is the need for further studies on a much wider range of species to establish the generality. Many well-aerated acidic soils have aqueous pH values >3.5 where although iron and manganese are readily detectable in the soil solution (see e.g. Table I), their solubility is low and toxicity is perhaps unlikely. However, the potential for iron and manganese toxicity is very great in waterlogged soils due to the reduction of Fe3+ and Mn3+ to Fez+ and Mn2+ as the result of microbial respiration. Tolerance here primarily results from the diffusion of oxygen through the intercellular air species to the root surface at a sufficient rate to maintain detoxification by oxidation of Fez+ and Mn2+.Characteristic plaques of ferric and manganic minerals accumulate on the roots of wetland plants as the end result of this process (Snowden and Wheeler, 1995).

THE CALCICOLECALCIFUGE PROBLEM REVlSlTED

I1

Total exclusion of iron and manganese from the plant is not an option because of their importance as essential elements, and plants exhibit a very wide range of element concentrations. For example, concentrations of manganese exceeding 30 000 p g g- have been reported (Jaffri and Heime, 1977) in Alyxia ruhricaulis (Baill.) Guillaumin and Maytenus bureaviana (Loes.) Loes from ultrabasic soils in New Caledonia whereas several crop species show symptoms of toxicity at less than I p g g - ' (see Woolhouse, 1983). As in the case of aluminium, this must reflect a range of different tolerance mechanisms.

'

C. BICARBONATE TOXICITY AND IRON DEFICIENCY

Bicarbonate ion toxicity and iron deficiency have both been implicated in the failure of calcifuges on calcareous soils. Woolhouse (1 966a,b) studied the effects of bicarbonate ions on the root growth and physiology of both calcicole and calcifuge grass species. He suggested that the reduced growth of calcifuge plants on calcareous soils could be attributed to bicarbonate in the soil solution. He demonstrated that bicarbonate inhibited the uptake and translocation of iron in the calcifuge species Deschatnpsia flexuosn but not in the calcicoles Arrhenatherum elatius or Koeleria macrantha (Ledeb.) Schultes. Deschampsia flexuosa root tips showed a markedly higher rate of dark fixation of carbon dioxide than the other species leading to the suggestion that disturbance of organic acid metabolism may be important in determining the observed growth effects. The relationship between bicarbonate-induced changes in growth and organic acid metabolism was investigated in later studies. Lee and Woolhouse (1 969a) showed that bicarbonate ions in the concentration range found in calcareous soils inhibited the root growth of calcifuges more than calcicoles and that the inhibitory effect was on cell elongation. The calcicole species Arrhenntherum elatius had a larger root organic acid content than the calcifuge Deschampsia flexuosa (>80%) (Lee and Woolhouse, 1969b). Exposure of plants to 10 mM HCO; for 24 h resulted in large increases in the organic acid, notably malate, content of both Deschanzpsiu flexuosu and Arrhenatherum elatius, although the increase was proportionately greater in the former. The seminal root growth of these species was affected in a similar manner by application of malate and bicarbonate to the rooting medium, suggesting that the bicarbonate-induced inhibition of Deschampsia jlexuosa was the result of a direct effect of malate accumulation on growth. (Arrhenutherum elatius was much less affected by either treatment.) Lee and Woolhouse (1971), utilizing [2-I4C]acetate,demonstrated a more complete compartmentation of respiratory and non-respiratory malate pools in roots of Deschampsia jlexuosa than in Arrhenatherutn elatius. Treatment of Deschatnpsia roots with bicarbonate following a pulse of [2-I4C]acetatereduced the I4CO2output and increased the labelling of the amino acid fraction suggesting that compounds which could not be utilized in the tricarboxylic acid (TCA) cycle were being diverted into amino acids. An interest in the effects of bicarbonate on iron uptake and transport arose because studies on crop species had shown that bicarbonate in solution could

12

J. A. LEE

produce symptoms of lime-induced chlorosis (see e.g. Wallihan, 1961). Although some calcicole species do show transient symptoms of lime-induced chlorosis on calcareous soils, studies have demonstrated that many calcifuge species are much more susceptible (see e.g. Grime and Hodgson, 1969). In some species the symptoms of chlorosis can be relieved by spraying an iron chelate onto the leaves suggesting that iron deficiency is the cause. Whether or not there is a direct causal effect of bicarbonate on iron uptake and utilization, iron availability is potentially a key limitation on plant growth in well-aerated calcareous soils. The solubility of Fe(OH), reduces markedly between pH 4 and 8, approximately 1000 times for each unit, suggesting that calcifuges are adapted to a relatively high availability of iron which predisposes them to iron deficiency and chlorosis on calcareous soils (Grime and Hodgson, 1969). Romheld (1987) suggested that there are only three ways in which plants can increase the solubility of iron in the rhizosphere: (1) enhancing the reduction of Fe” to Fez+, (2) lowering the rhizosphere pH, and ( 3 ) solubilizing sparingly soluble inorganic Fe3’ compounds by plant-produced chelating agents. Romheld further suggested that these processes are inducible under iron-limiting conditions, and suppressed when iron is available to limit toxicity. Presumably calcicole species apply one or more of these mechanisms fo avoid iron limitation on calcareous soils, and Romheld and Marschner (1986a) proposed that plants show one of two basic strategies. The first strategy involves three adaptive components: (1) an iron deficiency-induced enhancement of Fe3’ reduction to Fe’ ‘ at the root surface followed by uptake of Fez+;(2) proton extrusion promoting the reduction of Fe3+ to Fez’ and (3) the release of reducing and/or chelating substances by the roots (see e.g. Hether el al., 1984). Enhanced Fe3+ reduction is the main feature of this strategy as the result of the activation of reductases on or near the root surface. Increased reductase activities lead to the reduction of soluble organic Fe” to Fez’, the breakdown of iron chelates, and increased Fez+ uptake. Unlike the first strategy, which is widespread in higher plants, the second strategy is confined to grasses. It is characterized by an iron deficiency-induced release of Fe”-chelating molecules, the phytosiderophores (Takagi et al., 1984) coupled with a high affinity transport protein for Fe” phytosiderophores (Romheld and Marschner, 1986a). Romheld and Marschner (1986b) showed that Fe3+ phytosiderophores are absorbed as the entire chelate molecule and at high rates. Phytosiderophores include hydroxy- and amino-substituted immunocarboxylic acids, e.g. mugineic and avenic acids, and their release and uptake are only slightly depressed at high soil pH (Romheld and Marschner, 1986b). The uptake process is apparently highly specific for Fe” phytosiderophores as against synthetic or microbial iron chelates (Romheld and Marschner, 1986b), but the role of microbially mediated Fe” uptake in, for example, vesicular-arbuscular mycorrhizal grasses in calcareous soils should not be ignored. For example, some mycorrhizai fungi produce hydroxamate siderophores which are much more effective iron chelators than the low-molecular-weight organic acids secreted by plant roots.

13

THE CALCICOLE-CALCIFUGE PROBLEM REVISITED

TABLE I1 The production of shoot and rout dry mass by seven calcifuge species grown for 60-80 days

in a rendzic leptosol soil receiving supplemental iron or phosphate (From Tvler; 1994)

Dry weight (mg)

-P-Fe Cnrex pilulijera Deschnmpsia flexuosa Galium saxatile

Holcus mollis Luzuia pilosu Nurdus strictu Veronica ofJicinalis

171" 193" 122" 83"h 23" 7"

shoot root shoot root shoot root shoot root shoot root shoot root shoot root

85" 412" 221" 179" 139" 73" 172"

57"

fP-Fe

-P+Fe

464" 378"

112' 14 0 106 60

244" 99"

< I"

80

5 294'

24h

747"

305"

603'

256"

247" 214h 6 I" 348h 93b

163" 194" 83" 142'

76"

50"

Figures are means of 10 plants, means in each row carrying different superscript letters are significantly different ( P < 0.01).

D.

PHOSPHATE

Calcifuge seedlings grown on calcareous soils frequently show symptoms of phosphorus deficiency (see e.g. Lee and Woolhouse, 1966). Tyler (1992) and Tyler and Olsson (1993) demonstrated that calcifuge seedlings grew very poorly on calcareous soils, but growth was considerably improved by phosphate addition. In a later study (Tyler, 1994) adult individuals of seven perennial calcifuge species were transplanted onto a calcareous soil (pH-H208.1). The soil had either been left untreated or had been mixed with solid CaHPO, at the rate of 5 mol m-?. The soils were watered periodically with either distilled water or a 10 pM ferric citrate solution. Addition of CaHFQ, (but not iron) greatly enhanced the growth of all calcifuge species with the exception of Galium saxatile (Table 11). Iron supply was only shown to be the main limiting factor for Galium saxutile, and there was little evidence for the other species tested of a limitation in the uptake of iron from the calcareous soil. In fact Carex pilulifera and Veronica oficinalis showed reduced growth in response to iron addition. Plants grown in the unamended calcareous soil had exceptionally low phosphorus concentrations, pointing to the importance of phosphorus deficiency in most species as a major factor influencing the growth of calcifuges on calcareous soils. A similar growth response to iron and phosphorus supply in calcicole and calcifuge species was also observed in a UK rendzina soil (Davison, 1964, in Grime and Curtis, 1976).

14

J. A. LEE

5 1 e r and Strom (1995) examined the importance of organic acids in the acquisition of phosphorus and iron from calcareous soils. They investigated organic acid exudation in 10 calcifuge and 10 calcicole species. Calcicole species exuded three to four times more low-molecular-weight organic acids per unit of seed weight than calcifuges. The exudation of tricarboxylic acids was an order of magnitude greater for calcicole species, and the exudation of dicarboxylic acids was four to five times greater (Fig. 5). However, approximately three times more lactate was exuded from calcifuge than calcicole species. It was shown that the tricarboxylic citric acid and the dicarboxylic oxalic acid are powerful extractors of, respectively, iron and phosphate from calcareous soils whereas monocarboxylic acids are very ineffective extractors of either. p l e r and Strom (1995) conclude that the ability of calcicoles to extract phosphate and iron from calcareous soils through the exudation of di- and tricarboxylic acids is an important adaptive mechanism. However, some caution must be used in the generalization from laboratory exudation experiments on filter paper discs to the field where rhizosphere microorganisms may significantly modify exudates. Similarly other ions (e.g. bicarbonate) may have a significant and differential effect on the organic acid content (and possibly also exudation) of calcicole and calcifuge species (see above). Low phosphate availability is not confined to calcareous soils. In acidic soils the solubility of aluminium may limit phosphate availability through the formation of insoluble aluminium phosphates. An aluminium-stimulated synthesis and release of organic acids by calcifuge roots may increase phosphate availability through chelation. E. CALCIUM

In the last two decades, great advances have been made in our understanding of the role of calcium in plant growth and metabolism (see e.g. Marschner, 1994 for a review). In particular, the role of calcium as a second messenger (see e.g. McAinsh et al., 1990; Webb et al., 1996) has been widely recognized. This process depends on the maintenance of very low free Ca2+ concentrations in the cytosol (0.1-0.2 pM). Environmental signals activate membrane calcium channels (by e.g. depolarization of the plasma membrane), increasing calcium influx and cytosolic free Ca" concentrations. Calcium signals in the cytosol are targetted on calcium-dependent protein kinases which phosphorylate other enzymes and on calcium-binding proteins (calmodulins) (Roberts and Harmon, 1992). The biochemical properties of Ca2+ transporters in isolated plant membranes suggest that Ca2' efflux from the cytosol occurs through the action of ATPases localized at the plasma membrane and the tonoplast. In addition, a tonoplast Ca2+/nH+ antiporter may play a major role in Ca2' sequestration in the vacuole which is the major plant Ca2+ store (see Bush, 1995 for a review). The vacuole thus provides not only a sink, but also another potential source of stimulus-releasable Ca2+.If the role of calcium as a second messenger is universal in plant species as seems likely,

THE CALCICOLE-CALCIFUGE PROBLEM REVISITED

15

Formate

i

Acetate

Lactate

k

>0.05

30 01'

Succinate

+ malate Oxalate

Citrate

lsocitrate

<0.01"

Aconita te

I

I

1

0

0.5

1.o

Amount exuded (pmolg-1)

Fig. 5. Mean amounts (prnol g - ' seed) of low-molecular-weight organic acid anions exuded from species of limestone (n = 10) soils ( 0 )and acid silicate (n = 10) soils (@) and significance (P level) of the differences between means. Number of replicates of each species = 5 . From Tyler and Strom (1995).

16

J. A. LEE

the maintenance of very low cytosolic Ca2+ will be a key feature of adaptation to growth in calcareous soils. The differential response of calcicoles and calcifuges to calcium ions p e r se was clearly demonstrated by Jefferies and Willis (1964). The calcicole Origunum vulgare L. would not grow in sand culture watered with nutrient solutions containing less than 10 mg l-' calcium, and growth was stimulated up to 500 mg I-' Calcium. In contrast the calcifuge Juncus squarrosus L. could not survive calcium additions above 50 mg 1-' as either nitrate or chloride. A variety of mechanisms has been proposed to explain the effects of calcium on the growth of calcicoles and calcifuges (see e.g. Kinzel, 1983). However, a simple interpretation of the differential response of calcicoles and calcifuges is that adaptation to growth in calcareous soils has necessitated that the former minimize calcium influx through plasma membrane ion channels and maximize efflux and sequestration through membrane ATPases and the Ca2'/nH+ antiporter. Thus at low calcium supply, the calcium exclusion mechanisms in calcicoles effectively cause calcium deficiency. In contrast, calcifuges adapted to growth in soils of low calcium supply maximize calcium influx through plasma membrane ion channels (perhaps both calcium and potassium channels (see e.g. Bush, 1995)), but also have a lower efflux capacity from the cytosol than calcicoles which is overwhelmed at high calcium supply. This results in metabolically inhibitory concentrations in the cytosol and organelles. Effects of calcium supply on uptake processes, notably on potassium absorption, have been reported. The studies suggest that increased calcium supply stimulates potassium uptake in calcicoles but inhibits it in calcifuges (see. e.g. Olsen, 1942; Jefferies et al., 1969). Calcicoles show high tissue calcium concentrations, but it is doubtful whether more than a very small proportion of this is free Ca2+in solution in the cytoplasm, a view which is reinforced by the fact that calcicoles apparently have higher concentrations of calcium-binding proteins in the cytoplasm than calcifuges (Le Gales et al., 1980). Much of the calcium is probably sequestered in the vacuole as the soluble calcium malate or as the insoluble calcium oxalate. In some species appreciable calcium may be precipitated on leaf epidermal surfaces or cell walls as calcium carbonate (see e.g. Rorison and Robinson, 1984). However, there is still insufficient understanding of the localization of calcium in plants and the ways in which it might interfere with or determine cellular processes. Studies of stomatal behaviour in response to calcium supply are beginning to address these deficiencies. Ruiz et al. (1993) showed that the normal function of stomata in the calcifuge Lupinus luteus was affected by high calcium supply. Stomata1 opening in wellwatered plants was suppressed by high calcium supply, and mild water stress combined with high calcium supply led to excessive stomata1 closure and to a dramatic increase in calcium in the xylem sap. Elevated calcium levels affected photosynthetic capacity and water-use efficiency (Atkinson, 199I ; De Silva et al., 1994). However, the diffusive resistance of three calcicole species was unaffected by high concentrations (15 mol m-3) of calcium in the rhizosphere despite similarly

THE CALCICOLEXALCIFUGE PROBLEM REVISITED

17

high concentrations in the xylem sap. Nevertheless, stomata on isolated epidermis of two of the three species closed in response to free calcium above 1 mol m-' (De Silva and Mansfield, 1994). Thus the capacity to prevent most of the calcium in the xylem sap reaching the stomata1 guard cells may be a vital part of the adaptation of calcicoles to calcareous soils. De Silva et al. ( I 996) using X-ray microanalysis and a silver-rubeanate staining technique showed that in the leaves of Cerituurea scnbiosa L. and Leonrodon hispidus L. a substantial amount of the calcium is in the mesophyll, and much of that which enters the epidermis is found in the trichomes as calcium oxalate (Plate 1). Thus the calcium transport system in the cells at the base of calcium-rich trichomes may provide an important part of the overall adaptation of these calcicoles to calcareous soils, allowing calcium concentrations in the vicinity of the stomata to remain small thus ensuring that the essential role of Ca" in intracellular signalling i n guard cells can continue unimpeded. In plants which lack trichomes, other calcium localization mechanisms must operate. Ruiz and Mansfield (1994) showed that the only cells in the epidermis of mature Comnzelinu comtnunis leaves receiving protection from high levels of calcium are the guard cells. Protection is achieved by the very high content of calcium oxalate in the subsidiary cells of this species which must be receiving, and transporting through their cytosols, large amounts of free calcium. Calcium has an important structural role in plants, and also plays an important part in many physiological processes (see e.g. Hepler and Wayne, 1985) including as an intracellular second messenger (see above). To what extent the inability of calcifuges to exclude free calcium from guard cells represents the most important calcium toxicity mechanism requires further investigation. Similarly, the precise cause of the failure of calcicoles to grow on low concentrations of calcium requires further investigation at the cellular level, but may be primarily related to membrane transport processes, and differing affinities of plasma membranes in calcicole and calcifuge species for the Ca" ion. F. NITROGEN

The sensitivity of the nitrifying bacteria to low pH in culture combined with the predominance of ammonium in acidic soil solutions led to the conclusion that nitrate is unavailable in acid soils. This conclusion was supported by a number of laboratory studies which showed that calcifuges grew better on ammonium and calcicoles on nitrate as sole nitrogen sources (see e.g. Bogner, 1968; Gigon and Rorison, 1972). However, although nitrification may be absent from many acidic soils, particularly waterlogged ones, it is not absent from them all, and there is appreciable spatial and temporal variability in nitrate availability in acid soils (see e.g. Runge, 1974; Morecroft et ul., 1994). Nitrate utilization can be readily measured in situ by assays of the substrateinducible enzyme nitrate reductase. Such investigations have revealed that some calcifuge species utilize nitrate (see e.g. Havill er nl., 1974; Lee and Stewart, 1978; Gebauer et ul., 1988), and that in some circumstances nitrate utilization in acidic

18

J. A. LEE

soils may exceed that in shallow calcareous ones (Havill et al., 1977). However, many members of the Ericaceae show very low inducibility of nitrate reductase activity and have very low nitrate contents in the field (Havill et al., 1974; Gebauer et al., 1988). Thus there is apparently a wide variation in the ability of calcifuge species to utilize nitrate, although this variation may be reduced if their mycorrhizal fungi are effective in nitrate assimilation. The variation is much less in calcifuge bryophytes (see e.g. Morgan et al., 1992) reflecting the importance of the atmospheric reactive nitrogen supply to this group of plants. This source may also make an appreciated contribution (although frequently neglected) to the nitrogen economy of at least some higher plant calcifuge communities. There is as yet no unequivocal method of assessing the quantitative importance of ammonium assimilation to the nitrogen economy of calcicoles, but it would be surprising if this were not a significant nitrogen source in at least some calcareous soils (see e.g. Table I) given that incubation studies have shown up to 50% of mineralizable nitrogen as ammonium during winter months (Morecroft e f al., 1994; J. Carroll, personal communication). Interactions between the form of nitrogen available and other edaphic factors have been reported. Thus both Bogner (1968) and Gigon and Rorison (1972) demonstrated that calcifuges grew better on ammonium at low than high pH, that nitrate is toxic to these species at high pH, and that many calcicoles were unable to grow on ammonium at low pH. Rorison (1985) studied the interaction between aluminium and the form of available nitrogen on the growth of the calcifuge Deschampsia jexuosa, the calcicole Bromopsis erecta (Hudson) Fourr (Bromus erectus) and the wide-ranging species Holcus lanatus L. Aluminium (5.4 mg I-') had no adverse effect on the growth rate of Deschampsia and Holcus, most common on soils of intermediate pH, on ammonium at pH 4.5, but growth was reduced on nitrate (Fig. 6). The growth of Bromopsis was adversely affected by aluminium on both nitrogen sources, but growth on ammonium was poor both in the absence, and particularly in the presence, of aluminium. In the light of the findings by Calba and Jaillard (1997), it would be particularly interesting to see whether aluminium has differential effects on the uptake of nitrate and ammonium and the release of H+ in these species. The data may perhaps suggest that the inhibitory effect of aluminium was reduced by the species presumed major nitrogen source, but more work is required to establish this. The small effect of aluminium at 2.7mgI-' on Bromopsis growth on nitrate may perhaps be ascribed to the alkalization and precipation of aluminium at the root surface as a result of nitrate

Fig. 6. The main yields of individual plants of (a) Deschampsia Jexuosa, (b) Holcus and (c) Bromopsis erecta in response to different combinations of nitrogen and aluminium in a complete nutrient solution. The 95% C.L. are indicated where they exceed the dimension of the symbols. Only curves that differ significantly from each other ( P < 0.05) are. included. Open symbols, N03-N treatment; filled symbols, N&-N treatment. zero; 0, 2.7 mg I-'; X , 5.4 mg I-'. From Rorison (1985). Aluminium concentrations: (0) lanaius

19

THE CALCICOLE-CALCIFUGE PROBLEM REVISITED

( i i ) NH,-

( i ) NO,-N

N

( 0 )Desch,amps/o flexuosa

lorn[ I00

( b ) Holcus lonotus

3 1000-

c

A1

0

u u)

Y

al

Bromus crdctus

r A1 A1

0

20

30

40

0

l i m e (days)

20

30

40

20

J. A. LEE

uptake. These studies help to demonstrate the potential importance of interactions between edaphic factors which have largely been ignored in studies of the calcicole-calcifuge problem. The inorganic nitrogen pool represents only a very small fraction of the total nitrogen content of soils, and mineralization may limit plant growth in many semi-natural ecosystems. Utilization of at least a part of the organic nitrogen pool represents a potential way of avoiding this limitation. Mycorrhizal fungi degrade proteins in soil organic matter, absorbing and transferring amino acids to their host plants without nitrogen mineralization (Read, 1991), and are an important part of the adaptation of many calcifuge plants to growth in acidic soils. Evidence is accumulating that even non-mycorrhizal calcifuge plants are able to utilize amino acids (Chapin et al., 1993). Evidence is also accumulating that plants may be able to influence mineralization processes in soils directly. Northup et al. (1995) showed that the polyphenol concentrations in decomposing Pinus muricata litter control the proportion of nitrogen released as dissolved organic forms relative to ammonium and nitrate in soils of the Ecological Staircase in northern California. Pygmy forest communities on acidic nitrogen-limited soils produce litter rich in polyphenols resulting in high rates of dissolved organic nitrogen production relative to mineralized nitrogen (Fig. 7). These workers suggest that polyphenols mediate the enhanced mobilization of organic nitrogen, and that polyphenols in litter are higher on the more acid and infertile soils. They suggest that this feedback to soil conditions controls litter nitrogen mobilization in such a way as to facilitate nitrogen recovery by pine-mycorrhizal associations minimizing the availability of nitrogen to competing plants. Polyphenols in litter may also provide an important means of the inhibition of nitrification by calcifuges to a similar end (Baldwin et al., 1983). We still have too little knowledge of organic nitrogen utilization by plants and the ways in which plant-soil interactions may influence dissolved organic nitrogen release from litter to be able to make generalizations. However, this is potentially an important way in which some calcifuge species may be able to sequester nitrogen. This may be even more important given the paucity of species in acidic soils in temperate regions which have symbiotic associations with nitrogen-fixing microorganisms. Nodulation may be impaired by soil acidification either directly through the depression of the adsorption of Rhizobiurn at the root surface by for example high H ’ and low Ca” concentrations, or indirectly through changes in root morphology, root hair and lateral root formation (Caetono-Anolles et al., 1989). Soil acidity factors may also affect the ability of root exudates to induce the expression of nodulation genes (Richardson et al., 1988) and may limit the persistence of Rhizobium strains (Lowendorf and Alexander, 1983). G . MICROELEMENTS

There is little evidence to suggest that boron, cobalt or molybdenum deficiency plays a role in determining the distribution of calcicole and calcifuge species in

21

THE CALCICOLE-CALCIFUGE PROBLEM REVISITED

2

700

m

U

L

600 -

c

-

al a

500

I2

-2

400

L

300

m

.-

C

a Y

= 0)

200

Y

; 100

-

-

a

0

0 0

-

-

0

0.

0

0 0

a

0

m

Q

$

0

0,

?

c C

E"

200

-

100

-

500

v

2 400 I $ 300 m

a B C

al

m

2

.-c

z

-

600 - b

0

0 0

-

-

0

c

I

I

1

o

0

I

I

I

Fig. 7. Pinus rnuriculu litter nitrogen release versus concentration of total phenolics (a) dissolved organic nitrogen and (b) mineral nitrogen (NH: + NO;). Litter under monospecies clusters of pine was sampled in three contrasting soil acidity/fertility conditions on the Ecological Staircase, near Mendocino, California. From Northup et ul. (1995).

semi-natural ecosystems. The fact that nitrate reductase is a molybdenumcontaining enzyme suggests that molybdenum deficiency might limit nitrate utilization in calcifuge plants. However, experiments involving molybdenum fertilizer addition to calcifuge plants in situ have not induced further nitrate reductase activity in the presence of nitrate (Lee and Stewart, 1978). Grime and Curtis ( 1976) present data of A. W. Davison from a laboratory growth experiment in which a calcareous soil had been subjected to a range of nutrient amendments, including one involving the addition of potassium, magnesium, manganese and boron. The growth of calcifuge and calcicole seedlings in this treatment was similar to the control suggesting that not only the supply of microelements such as boron are not important determinants of seedling growth in calcareous soils, but also potassium and magnesium are also not limiting.

22

J. A. LEE

IV. CONCLUSION Recent work suggests that organic acid metabolism may play a key role in aluminium tolerance in calcifuges (Delhaize, 1993b), in the phosphorus and iron acquisition by calcicoles (51er and Strom, 1995), and in calcium regulation in the vicinity of guard cells (Ruiz and Mansfield, 1994), and thus may be pivotal to our understanding of plant adaptation to growth in acidic and calcareous soils. This pivotal position is further emphasized by the fact that bicarbonate is known to markedly perturb organic acid metabolism (Lee and Woolhouse, 1969a, 197 l), and assimilation of nitrate, particularly in the shoot results in the accumulation of organic acid anions as part of cellular pH regulation, and assimilation of ammonium in the roots depletes organic acid pools in amino acid synthesis (Raven and Smith, 1976). However, study of organic acid metabolism other than on CAM (Crassulacean Acid Metabolism) plants has been a deeply unfashionable area of physiological ecology for at least two decades, and there is little evidence of the controls on organic acid synthesis and exudation and how these might be influenced by edaphic factors. Delhaize er al. (1993b) provide evidence to suggest that malate synthesis and exudation in wheat is specifically controlled by aluminium, and is unaffected by other elements. A later study by this group (Ryan et al., 1995) showed that malate efflux was accompanied by potassium efflux to maintain electroneutrality, and that aluminium stimulates malate efflux via anion channels. There is the need to establish whether this effect is widespread in calcifuge species, and whether it is absent from strict calcicoles. A good starting point for a comparison between calcicoles and calcifuges would be an examination of the Anfhoxanthum odoratum ecotypes from the Park Grass experiment studied by Davies and Snaydon (1973). There is also the need to establish the specificity of the aluminium stimulation of malate synthesis by the use of a wider range of elements, and to establish the mechanism of stimulation. Ryan er al. (1995) showed that the greater malate efflux from aluminium-tolerant than aluminium-sensitive wheat plants could not be explained by differences in the activities of phosphoenol pyruvate (PEP) carboxylase and NAD-malate dehydrogenase. A possible model which might go some way to explain the involvement of organic acid metabolism in adaptation to growth in acidic and calcareous soils is represented in Fig. 8. In this model, primary control over anion channels is accorded to A13+ in calcifuges and to Ca2+ in calcicoles. When seedlings of calcicoles and calcifuges are grown in acidic soils A13+is absorbed less strongly by calcifuge than calcicole root plasma membranes, ungating anion channels in the former, allowing the release of malate and citrate into the apoplast and rhizosphere which reduces the uptake of A13+.The continued release of malate and citrate into the apoplast is achieved by continual replenishment of the cytosolic pool either by metabolism or by the controlled release of the vacuolar pool. In contrast, A13+ is more strongly absorbed by calcicole root plasma membranes and their anion channels are not ungated. Uptake of A13+into the cytosol leads to toxicity and may directly reduce malate synthesis through the destruction of oxaloacetate. Any

23

THE CALCICOLE-CALCIFUGE PROBLEM REVISITED

Acidic Soils ( High A13'in soil solution ) Apoplast

w 1 1 - 1

4

Al Malate

Al

Apoplast Al

Cytosol

I

-1.1

Cytosol At

-

AI Al-

Malate

A1

Malate .......................

)

Al(Al decomposes O M and

rsducas malate pool 1

Al-

Al

+

AlCitrate

Al

Citrate

Al

At

E...

Citrate ....................

-

AlAl

I

Calcicole root plasma membrane

Calcifuge root plasma membrane

Calcareous Soils ( High Ca2+ and HCOQin soil solution ) Apoplast

Apoplast

Cytosol

L

q

c

Cytosol

a Ca

("

.............................

i

CaCa Ca

-

CaCa

r

Make Citrate

-

Ca-

-

HC03

Ca

Malate Citrate

Malate

-................... __..:

.

PH

4

Fe citrate

Ca

Calcifuge root plasma membrane

Ca Calcicole root plasma membrane

Fig. 8. A model of the effects of A13+ and Ca" on plasma membrane and cytosolic processes in calcicole and calcifuge roots in acidic and calcareous soils. Solid arrows indicate strong net fluxes, dotted arrows indicate little or no net fluxes. The size of the malate and citrate typeface indicates the relative size of their pools in the apoplast and cytosol.

24

1. A. LEE

reduction in organic acid synthesis would reduce the potential for limiting aluminium toxicity through chelation either in the cytosol or in the apoplast. When seedlings of calcicoles are grown in calcareous soils, a rise in cytosolic Ca2+ results in the ungating of anion channels in the plasma membrane and a stimulation of organic acid synthesis further enhanced by bicarbonate uptake. The release of organic acids into the apoplast and rhizosphere enhances iron and phosphorus uptake through chelation processes. Fe3+ is reduced to Fe2+ at the plasma membrane surface and the latter is transported into the cytosol. In contrast, calcifuge species show poor control over cytosolic Ca2+concentrations in their root cells, and anion channels in the plasma membranes remain largely gated. This results in the accumulation of organic acids in the cytosol with possible disruption of pH, a process which may be exacerbated if rising cytosolic Ca2+ affects the release of organic acids from the vacuole. This model depends heavily on differential controls of plasma membrane anion channels in calcicoles (by Ca2+)and calcifuges (by Al'+). There is, however, as yet insufficient evidence in its support, or to assess the importance of effects of these ions on tonoplast processes in calcicoles and calcifuges. Molecular biological techniques have so far had little influence on our understanding of the calcicole-calcifuge problem, but have great potential. For example, it should be possible to transform calcifuge plants to express an unregulated PEP carboxylase to show enhanced synthesis and exudation of di- and tricarboxylic acids. It might thus be possible to establish the controls on organic acid synthesis and exudation, and the relative importance of phosphate and iron acquisition to the overall adaptation to growth in calcareous soils. It should also be possible to use these techniques to explore more fully the aluminium stimulation mechanism for malate synthesis as proposed by Delhaize and Ryan (1995). Similarly, it should be possible to transform plants to overexpress nitrate and nitrite reductase activities (see e.g. Scheible et af. (1997). This might be particularly interesting in the Ericaceae where nitrate assimilation in many species is very low (see e.g. Lee and Stewart, 1978), and where enhanced nitrate assimilation ability might influence the ability of these species to compete for example with calcifuge grasses on nitrifying acidic soils and to grow on calcareous soils. Another modem technology which has not been widely employed in an ecological context is the use of the patch clamp technique to study ion relations. There is still much to be learnt particularly about the role of calcium in the regulation of ion transport processes, and how this may vary between calcicole and calcifuge species. The importance of calcium in maintaining membrane integrity, and in influencing the uptake and toxicity of many other elements including iron and manganese (see e.g. Foy et ol., 1978) strongly suggests the need for much further study. There is the need to improve our knowledge of the influence of calcium ions at the intracellular level in calcicoles and calcifuges, and their part in signal transduction events other than in stomata1 guard cells. Similarly in viva NMR techniques may be useful in understanding the location and function of K + , NO, NHd and malate pools, and how these are affected by edaphic factors (see e.g.

THE CALCICOLE-CALCIFUGE PROBLEM REVISITED

25

Lee and Ratcliffe, 1993).In vivo NMR has shown that the bulk of aluminium in tea leaves is present as a soluble Al-catechin complex (Nagata et ul., 1992). The proposed importance of processes at or near root surfaces in determining adaptation to growth in acidic and calcareous soils also brings in to focus the role of mycorrhizal and rhizosphere microorganisms. The key role that the former play in phosphorus acquisition has long been recognized, and if the failure of calcifuges on calcareous soils can simply be ascribed to phosphorus deficiency (Tyler and Olsson, 1993), it is tempting to conclude that this may primarily reflect effects on the mycorrhizal fungi. If calcicole plants do show high di- and tricarboxylic acid exudation on calcareous soils as a mechanism to enhance phosphorus and iron acquisition, this must influence at least to some extent rhizosphere microorganisms. The ability to enhance further this exudation by the use of transgenic plants raises the possibility of examining the quantitative effects of the exudates on microbial populations. Although larger rates of exudation might increase both microbial populations and their competitive ability to absorb phosphate, it may also result in increases in the activity of phosphatases in the rhizosphere and hence greater overall phosphate availability. The success of the proposed malate/citrate aluminium detoxification mechanism will depend critically on whether rhizosphere microorganisms are able to metabolize organic acids rapidly. At least as far as the bacterial component of the rhizosphere is concerned, it should be possible to screen the root systems of a wide range of calcifuge species using the BIOLOG plate technique to see whether their associated bacteria are readily capable of utilizing malate as a sole carbon source. Rapid scrcening of the rhizosphere fungal populations must await improvement in existing technology. The difference between the vegetation of acidic and calcareous soils remains on the one hand one of the most important and readily made observations in plant ecology, and on the other still one which is not entirely understood. For too long in this field physiological ecologists have relied on inferences from studies of crop plants which may not be readily applicable to adaptations to the more extreme acidic and calcareous soils. The challenge facing physiological ecologists now is to answer the outstanding questions by the application, at least in part, of modem molecular techniques to the study of important calcicole and calcifuge species.

ACKNOWLEDGEMENTS I gratefully acknowledge the many helpful comments and suggestions made by J. R. Leake, R. C. Leegood and M. C. Press on an earlier version of this paper. I also thank J. R. h a k e for permission to present his soil chemistry data for Coombesdale, Derbyshire.

REFERENCES Andersson, M. E. and Brunet, J. (1993). Sensitivity to H and A1 ions limiting growth and distribution of the woodland grass Brornus benekenii. P lntit crrid Soil 153, 243-254.

26

J . A. LEE

Aniol, A. and Gustafson, J. P. (1984). Chromosome location of genes controlling aluminum tolerance in wheat, rye and triticale. Canadian Journal of Genetics and Cytology 26, 701-705. Arnon, D. I. and Johnson, C. M. (1942). The influence of hydrogen ion concentration on the growth of higher plants under controlled conditions. Plant Physiology 17, 525-539. Atkinson, C. J. (1991). The flux and distribution of xylem sap calcium to adaxial and abaxial epidermal tissue in relation to stornatal behaviour. Journal of Experimental Botany 42, 987-993. Baldwin, I. T., Olson, R. K. and Reiners, W. A. (1983). Protein binding phenolics and the inhibition of nitrification in subalpine Balsam fir soils. Soil Biology and Biochemistry IS,419423. Bartlett, R. J. and Riego, D. C. (1972). Effect of chelation on the toxicity of aluminium. Plant and Soil 37,419-423. Bogner, W. (1968). Experimentelle Priifung von Waldbodenpflanzen auf ihre Anspriiche an die Form der Stickstofferniihrung. Mitteilungen des Vereins fur Forstliche Standortskunde und Forstpflanzenzuchtung 1 8 9 , 3 4 5 . Bush, D. S. (1995). Calcium regulation in plant cells and its role in signaling. Annual Review of Plant Physiology and Molecular Biology 46, 95-122. Caetono-Anolles, G., Lagares, A. and Favelukes, G. (1989). Adsorption of Rhizobium meliloti to alfalfa roots: dependence on divalent cations and pH. Plant and Soil 117, 67-74. Calba, H. and Jaillard, B. (1997). Effect of aluminium on ion uptake and H+ release by maize. New PhytoZogist 137,607416. Cameron, R. S., Ritchie, G. S. P. and Robson, A. D. (1986). Relative toxicities of inorganic aluminum complexes to barley. Soil Science Sociefy of America Journal 50, 12311236. Caradus, J. R. and Mackay, A. D. (1995). Distribution of Al-tolerance in crosses between genotypes of white clover selected for either Al-tolerance or Al-susceptibility, In “Plant Soil Interaction at Low pH” (R. A. Date, N. J. Grundon, G. E. Raymont and M. E. Probert, eds) pp. 447-450. KIuwer Academic Publishers, Dordrecht. Chapin 111, F. S.,Mollanen, L. and Kielland, K. (1993). Preferential use of organic nitrogen for growth by a non-mycorrhizal arctic sedge. Nature 361, 150-153. Christiansen-Weniger, C., Groneman, A. F, and van Veen, J. A. (1992). Associative N2fixation and root exudation of organic acids from wheat cultivars of different aluminium tolerance. Plant and Soil 139, 167-174. Clarkson, D. T. (1966). Aluminium tolerance in species within the genus Agrostis. Journal of Ecology 54,167-178. Davies, M. S. and Snaydon, R. W. (1973). Physiological differences among populations of Anthoxanthum odoratum L. collected from the Park Grass experiment, Rothamsted. 11. Response to aluminium. Journal of Applied Ecology 10, 47-55. Delhaize, E. and Ryan, P. C. (1995). Aluminum toxicity and tolerance in plants. Plant Physiology 107 3 15-32 1. Delhaize, E., Craig, S., Beaton, C. D., Bennett, R. J., Jagadish, V. C. andRandall, P. J. (1993a). Aluminum tolerance in wheat (Triticum aestivum L.). I. Uptake and distribution of aluminum in root apices. Plant Physiology 103, 685-693. Delhaize, E., Ryan, P. R. and Randall, P. J. (1993b). Aluminum tolerance in wheat (Triticum aestivum L.). 11. Aluminum-stimulated excretion of malic acid from root apices. Plant Physiology 103, 695-702. De Silva, D. L. R. and Mansfield, T. A. (1994). The stomatal physiology of calcicoles in relation to calcium delivered in the xylem sap. Proceedings of the Royal Society, London B 257,81-85. De Silva, D. L. R., Ruiz, L. R., Atkinson, C. J. and Mansfield, T. A. (1994) Physiological disturbance caused by high rh,izosphere calcium in the calcifuge. Lupinus luteus.

THE CALCICOLE-CALCIFUGE PROBLEM REVISITED

27

Journal of Experimental Botany 45, 585-590. De Silva, D. L. R., Hetherington, A. M. and Mansfield, T. A. (1996). Where does all the calcium go? Evidence of an important regulatory role of trichomes in two calcicoles. Plant, Cell and Environment 19, 880-886. Falkengren-Grerup, U. and Tyler, G. (1 993). Soil chemical properties excluding field-layer species from beech forest mor. Plant and Soil, 148, 185-19 1. Foy, C. D., Chaney, R. L. and White, M. C. (1978).The physiology of metal toxicity in plants. Annual Review of Plant Physiology 29, 5 11-566. Gassmann, W. and Schroeder, J. I. (1994). Inward-rectifying K+ channels in root hairs of wheat. A mechanism for aluminum-sensitive low affinity K* uptake. Plant Physiology 105, 1399-1408. Gebauer, G., Rehder, H. and Wollenweber, B. (1988). Nitrate, nitrate reduction and organic nitrogen in plants from different ecological and taxonomic groups of Central Europe. Oecologia 75, 371-385. Gigon. A. and Rorison, I. H. (1972). The response of some ecologically distinct plant species to nitrate and to ammonium-nitrogen. Journal of Ecology 60, 93-102. Grime, J. P. and Curtis, A. V. (1976). The interaction of drought and mineral stress in calcareous grassland. Journal of Ecology 64, 975-988. Grime, J. P. and Hodgson, J. G. (1969). An investigation of the ecological significance of lime-chlorosis by means of large-scale comparative experiments. I n “Ecological Aspects of the Mineral Nutrition of Plants” (I. H. Rorison, ed.) pp. 357-380. Blackwell Scientific Publications, Oxford. Havill, D. C., Lee, J. A. and Stewart, G . R. (1974). Nitrate utilization by species from acidic and calcareous soils. New Phytologist 73, 1221-1231. Havill, D. C., Lee, J. A. and De Felice, J. (1977). Some factors limiting nitrate utilization in acidic and calcareous grasslands. New Phytologist 78, 649-659. Hepler, P. K. and Wayne, R. 0. (1985). Calcium and plant development. Annual Review of Plant Physiology and Plant Molecular Biology 36, 397-439. Hether, N. H., Olsen, R. A. and Jackson, I. L. (1984). Chemical identification of iron reductants exuded by plant roots. Journal of Plant Nutrition 7 , 667-676. Hewitt, E. J. (1952). A biological approach to the problems of soil acidity. Transactions ofthe International Society of Soil Science Joint Meeting, Dublin 1, 107-118. Hope-Simpson, J. (1938). A chalk flora of the Lower Greensand: its uses in interpreting the calcicole habit. Journal of Ecology 26, 218-235. Hunter, D. and Ross, D. S. (1991). Evidence for a phytotoxic hydroxy-aluminum polymer in organic soil horizons. Science 251, 10561058. JaffrC, T. and Heime, R. (1977). Accumulation du manganese par des espkes associCs aux terrain ultrabasiques de Nouvelle-Caledonie. Compte Rendu del’Acadkmie des Sciences Series D 284, 1573-1575. Jefferies, R. L. and Willis, A. J. (1964). Studies on the calcicole-calcifuge habit. 11. The influence of calcium on the growth and establishment of four species in soil and sand cultures. Journal of Ecology 52, 691-707. Jefferies, R. L., Laycock, D., Stewart, G. R. and Sims, A. P. (1969). The properties of mechanisms involved in the uptake and utilization of calcium and potassium by plants in relation to an understanding of plant distribution. In “Ecological Aspects of the Mineral Nutrition of Plants” (I. H. Rorison, ed.) pp. 281-307. Blackwell Scientific Publications, Oxford. Johnson, P. A. and Bennett, R. J. (1991). Aluminium tolerance of root cap cells. Journal of Plant Physiology 137, 760-762. Jones, D. L. and Kochian, L. V. (1995). Aluminum inhibition of the inositol 1,4,5-triphosphate signal transduction pathway in wheat roots: a role in aluminum toxicity? Plant Cell 7 , 1913-1 922.

28

J. A. LEE

Jones, D. L., Kochian, L. V. and Gilroy, S. (1998). Aluminum induces a decrease in cytosolic calcium concentration in BY-2 tobacco cell cultures. Plant Physiology 116, 8 1-89. Kinraide, T. B. (1991). Identity of the rhizotoxic aluminium species. Plant and Soil 134, 167-1 78. Kinraide, T. B. and Parker, D. R. (1990). Apparent phytotoxicity of mononuclear hydroxyaluminium to four dicotyledonous species. Physiologia Platitarum 79, 283-288. Kinraide, T. B. and Ryan, P. R. (1991). Cell surface charge may obscure the identity of the rhizotoxic aluminum species. In “Current Topics in Plant Biochemistry and Physiology 1991”. Ultraviolet-B Radiation Stress, Aluminum Stress, Toxicity and Tolerance, Boron Requirements, Stress and Toxicity. Vol. 10 (D. D. Randall, D. G. Blevins and C. D. Miles, eds) pp. 94-1 06. Interdisciplinary Plant Biochemistry and Physiology Program, University of Missouri-Columbia, USA. Kinzel, H. (1983). Influence of limestone, silicates and soil pH on vegetation. In “Physiological Plant Ecology 111. Responses to the Chemical and Biological Environment”, Encyclopedia of Plant Physiology, New Series, Vol. 12c (0. L. Lange, P. S. Nobel, C. B. Osmond and H. Ziegler, eds) pp. 201-244. Springer-Verlag, Berlin. Kochian, L. V. (1995). Cellular mechanisms of aluminum toxicity and resistance in plants. Annual Review of Plant Physiology and Molecular Biology 46, 237-260. Lee, J. A. and Woolhouse, H, W. (1966). Chlorophyll content of Deschampsia ,flexuostr seedlings grown on a calcareous and non-calcareous soil. Nature 209, 1044-1045. Lee, J. A. and Woolhouse, H. W. (1969a). A comparative study of bicarbonate inhibition of root growth in calcicole and calcifuge grasses. New Phytologist 68, 1-14. Lee, J. A. and Woolhouse, H. W. (1969b). Root growth and dark fixation of carbon dioxide in calcicoles and calcifuges. New Phyrologist 68, 247-255. Lee, J. A. and Woolhouse, H. W. (1971). The relationship of compartmentation of organic acid metabolism to bicarbonate ion sensitivity of root growth in calcicoles and calcifuges. New Phytologist 70, 103-1 I 1. Lee, J. A. and Stewart, G. R. (1978). Ecological aspects of nitrogen assimilation in plants. Advances in Botanical Research 6, 1-40. Lee, R. B. and Ratcliffe, R. G. (1993). Nuclear magnetic resonance studies of the location and function of plant nutrients in vivo. Plant and Soil 155, 45-55. Le Gales, Y. Lamart, A. and Heller, R. (1980) Fixation du calcium par des fractions macromoleculaires solubles isolCes a partir de vCgktaux supkrieurs. Physiologie Vkgetale 18, 43 1-441. Lowendorf, H. S. and Alexander, M. (1983). Identification of Rhizobium nhaseoli strains that are tolerant or sensitive to soil acidity. Applied Environmental Microbiology 45, 737-742. McAinsh, M. R., Brownlee, C. and Hetherington, A. M. (1990). Abscisic acid-induced elevation of guard cell cytosolic Ca2+ precedes stornatal closure. Nature 343, 186-1 88. Mahmoud, A. and Grime, J. P. (1977). A comparison of the susceptivility of Arrhenarherum elatius (L.) Beau ex J. C.Presl., Agrostis tenuis Sibth., DeschampLsiafIexuosu and Festuca ovina L. to manganese toxicity. Plant arid Soil 47, 559-565. Marschner, H. (1995) “Mineral Nutrition of Higher Plants”. Academic Press, London. Morecroft, M. D., Sellers, E. K. and Lee, J. A. (1994). An experimental investigation into the effects of atmospheric nitrogen deposition on two semi-natural grasslands. Journal of Ecology 82, 475-483. Morgan, S. M., Lee, J. A. and Ashenden, T. W. (1992). Effects of nitrogen oxides on nitrate assimilation in bryophytes. New Phytologist 120, 89-97. Nagata, T., Hayatsu, M. and Kosuge, N. (1992). Identification of the aluminium forms in tea leaves by ’7AI NMR. Phytochenzistry 31, 1215-1218. Northup, R. R., Zengshou, Y., Dahlgren, R. A. and Vogt, K. A. (1995). Polyphenol control

THE CALCICOLE-CALCIFUGE PROBLEM REVISITED

29

of nitrogen release from pine litter. Mature 377, 227-229. Olsen, C. (1942). Water culture experiments with higher green plants in nutrient solutions having different concentrations of calcium. C.R. Laboratory Curlsberg, Series Chimica 24, 69-98. Papernik, L. A. and Kochian, L. V. (1997). Possible involvement of Al-induced electrical signals in Al tolerance in wheat. Plant Physiology 115, 657-667. Pigott, C. D. (1962). Soil formation on carboniferous limestone of Derbyshire, I. Parent materials. Journal of Ecology 50, 145-156. Pineros, M. and Tester, M. (1993). Plasma membrane Ca” channels in roots of higher plants and their role in aluminium toxicity. Plant and Soil 155/156, 119-122. Puthota, V., Gug-Ortega, R., Johnson, J. and Ownby, J. (1991). An ultrastructural study of the inhibition of mucilage reaction in the wheat root cap by aluminium. In “Plant-Soil Interactions at Low pH” (R. J. Wright, V. C. Baligar, R. P. Murrmann, eds) pp. 779-787. Kluwer Academic, Dordrecht. Raven, J. A. and Smith, F. A. (1976). Nitrogen assimilation and transport in vascular land plants in relation to intracellular pH regulation. New Phyrologisr 76, 41 5 4 3 1 . Read, D. J. (1991). Mycorrhizas in ecosystems. Experienria 47, 376-391. Reid, D. A. (1971). Genetic control of reaction to aluminium in winter barley. In “Barley Genetics 11. Proceedings 2nd International Barley Genetics Symposium, Pullman, Washington” (R. A. Nilan, ed.). Washington State University Press, Pullman. Richardson, A. E., Djordjevic, M. A,, Rolfe, B. G. and Simpson, R. J. (1988). Effects of pH, Ca and A1 on the exudation from clover seedlings of compounds that induce the expression of modulation genes in Rhizobium trifolii. Plant and Soil 109, 3 7 4 7 . Ritchie, G. S. P. (1994). The role of dissolution and precipitation of minerals controlling soluble aluminium in acidic soils. Advances in Agronomy 53, 47-83. Roberts, D. M. and Harmon, A. C. (1992) Calcium-modulated proteins: targets of intracellular calcium signals in higher plants. Annual Review of Plant Physiology and Molecular Biology 43, 3 7 5 4 14. Romheld, V. (1987) Different strategies for iron acquisition in higher plants. Physiologiu Planfarum 70, 23 1-234. Rijmheld, V. and Marschner, H. (1986a). Mobilization of iron in the rhizosphere of different plant species. Advances in Plant Nutrition 2, 1.55-204. Romheld, V. and Marschner, H. (1986b). Evidence for a specific uptake system for iron phytosiderophores in roots of grasses. Plant Physiology 80, 175-1 80. Rorison. I. H. (1960a). Some experimental aspects of the calcicole-calcifuge problem. I. The effects of competition and mineral nutrition upon seedling growth in the field. Journal of Ecology 48, 585-599. Rorison, I. H. (1960b). The calcicole-calcifuge problem 11. The effects of mineral nutrition in seedling growth in the field. Journal of Ecology 48, 679-688. Rorison, I. H. (1985). Nitrogen source and the tolerance of Descbampsia flexuosu, Holcus lanatus and Bromus erectus to aluminium during seedling growth. Journal of Ecology 73, 83-90. Rorison, 1. H. and Robinson, D. (1984). Calcium us an environmental variable. Plunt, Cell cmd Environment 7, 38 1-390. Ruiz, L. P., Atkinson, C. J. and Mansfield, T. A. (1993). Calcium in the xylem and its influence on the behaviour of stomata. Philosophical Tran.sac>tionsof rhe Royul Sociey of London, B 341, 67-74. Ruiz, L. P. and Mansfield, T. A. (1994). A postulated role of calcium oxalate in the regulation of calcium ions in the vicinity of stomata1 guard cells. New Phytofogist 127. 473481. Runge, M. ( 1974). Die Stickstoff-Mineralisation im Boden eines Sauerhumus-Buchenwaldes. 11. Die Nitratproduktion. Oecologiu Plunrrrrum 9, 219-230.

30

J. A. LEE

Ryan, P. R., Delhaize, E. and Randall, P. J. (1 995). Characterisation of Al-stimulated efflux of malate from the apices of Al-tolerant wheat roots. Planta 196, 103-110. Scheible, W-R., Gonzalez-Fontes, A., Morcuende, R., Lauer, M., Geiger, M., Glaab, J., Gajon, A., Schulze, E-D. and Stitt, M. (1997). Tobacco mutants with a decreased number of functional nia genes compensate by modifying the diurnal regulation of transcription, post-translational modification and turnover of nitrate reductase. Planta 203, 304-319. Snowden, R. E. D. and Wheeler, B. D. (1995). Chemical changes in selected wetland plant species with increasing iron supply, with specific reference to root precipitates and iron tolerance. New Phytologist 131, 503-520. Takagi, S., Nomoto, K. and Takemoro, T. (1984). Physiological aspects of mugeneic acid a possible phytosiderophore of graminaceous plants. Journal of Plant Nutrition 7 , 469471. Tansley, A. G.(1917). On competition between Galiurn saxatile L. (G. hercynicurn Weig) and G. sylvestre Poll (G. asperurn schreb) on different types of soil. Journal of Ecology 5, 173-179. Taylor, G. (1995). Overcoming barriers to understanding the cellular basis of aluminium resistance. In “Plant Soil Interactions at Low pH” (R. A. Date, N. J. Grundon, G. E. Rayment and M. E. Probert, eds) pp. 255-269. Kluwer Academic Publishers, Dordrecht . Tyler, G. (1992). Inability to solubilise phosphate in limestone soils - key factor controlling calcifuge habit of plants. Plant and Soil 145, 65-70. Tyler, G. (1994). A new approach to understanding the calcifuge habit of plants. Annals of Botany 73, 321-330. Tyler, G. and Olsson (1993). The calcifuge behaviour of Viscaria vulgaris. Journal of Vegeration Science 4, 29-36. Tyler, G.and Strom, L. (1995). Differing organic acid exudation patterns explain calcifuge and acidifuge behaviour of plants. Annals of Botany 75, 75-78. Wallihan, E. F. (1961). Effects of sodium bicarbonate on iron absorption by orange seedlings. Plant Physiology 36, 52-53. Webb, A. A. R., McAinsh, M. R., Taylor, J. E. and Hetherington, A. M. (1996). Calcium ions as intracellular second messengers in higher plants. Advances in Botanical Research 22, 45-96. Wieland, E. and Stumm, W. (1992). Dissolution kinetics of kaolinite in acidic aqueous solutions at 25OC. Geochimica Cosmochimica Acta 56, 3339-3355. Woolhouse, H. W. (1966a). Comparative physiological studies on Descharnpsia fiexuosa, Holcus mollis, Arrhenatherum elatius and Koeleria gracilis in relation to growth on calcareous soils. New Phyrologist 65, 22-3 1. Woolhouse, H. W. (1966b). The effect of bicarbonate on the uptake of iron in four related grasses. New Phytologist 65, 372-375. Woolhouse, H. W. (1969). Differences in the properties of the acid phosphatases of plant roots and their significance in the evolution of edaphic ecotypes. In “Ecological Aspects of the Mineral Nutrition of Plants” (I. H. Rorison, ed.) pp. 357-380. Blackwell Scientific Publications, Oxford. Woolhouse, H. W. (1983). Toxicity and tolerance in the responses of plants to metals. In “Physiological Plant Ecology 111. Responses to the Chemical and Biological Environment”. Encyclopedia of Plant Physiology New Series, 12C (0. L. Lange, P. S. Nobel, C. B. Osmond and H. Ziegler, eds) pp. 245-300. Springer-Verlag. Berlin. Yermiyahu, U., Brauer, D. K. and Kinraide, T. B. (1997). Sorption of aluminum to plasma membrane vesicles isolated from roots of Scout 66 and Atlas 66 cultivars of wheat. Plant Physiology 115, 1119-1125.

Ozone Impacts on Agriculture: An Issue of Global Concern

M. R. ASHMORE and F. M. MARSHALL

Centre for Environmental Technology, Imperial College of Science Technology and Medicine, Silwood Park, Ascot, Berks SL5 7PI: UK*

I.

Introduction .........................................................................................................

11. Ozone Impacts on Agricultural Crops ..... ....... ............................. A. Exposure-Response Studies .......................................................................

32 33 34

111. Rural Ozone Levels in Developing Countries ........

36

IV. Direct Evidence of Adverse Effects on Crops ._...... A. Studies with Field Chambers ...................................................................... B. Studies with Ozone Protectant Chemicals ..................................................

39 39 41

V.

Responses to Ozone of Tropical Crops and Cultivars ....................................... A. Experimental Studies ................................................................... ................ . B. Factors Influencing Ozone Sensitivity in the Field ....................................

V1. Future Concentrations and Impacts of Ozone .................................................... VII. Conclusions ............................................ Acknowledgements .. References ................

43 43 45

46 46 48 49

While ozone has been shown to be the most important air pollutant affecting national crop production in North America and western Europe, mainly because it is found at phytotaxic concentrations over large areas, its impact in the developing countries qf Asia, Africa and Latin America, where the economic and social consequences of loss of production may be much greatec is uncertain. This review assesses the current and future significance c$ ozone impacts on agriculture in these countries. Although the information available on rural ozone *Address for correspondence: Department of Environmental Science, University of Bradford, Bradford, W. Yorks, BD7 1DP Advances in Botanical Rexarch Vol. 29

incorporating Advance, in Plant Psthulogy ISBN 0-12-11059?Y-0

Copynghi 0 19YY Academic Press All rigtits o l reproduction in any lurm reserved

32

M. R. ASHMORE and F. M . MARSHALL

concentrations is very limited, it does show that these concentrations can be high enough to have adverse effects on sensitive species, while a limited number of experimental studies have shown decreases in yield of staple crops due to ambient ozone. Current projections oj' rising emissions of ozone precursors suggest that the impacts on agriculture may increase very rapidly over the next two decades. There is an urgent need for more rural studies to determine ozone concentrations, and their impact on major crop species. in order to assess the current scale of the problem, and to develop models to estimate the future impacts of increased emissions.

I. INTRODUCTION The impacts of air pollution have long been recognized as an issue for concern in agriculture both in North America and Europe. Research programmes in both continents have provided extensive information on the physiological effects of pollutants, the relative sensitivity of different crop species and cultivars, and the air pollutant concentrations at which adverse impacts are found (e.g. Heck et al., 1988; Jager et al., 1993). Attempts have also been made to quantify the impacts of different pollutants on crop yields at national or regional levels (Adams et a/., 1988; van der Eerden et al., 1988). These assessments consistently indicate that, although pollutants such as sulphur dioxide and particulates may have significant local impacts on crop production, the air pollutant which is clearly the most important in terms of regional or national economic impacts on agriculture is ozone. Ozone is not emitted directly into the atmosphere. Instead, high atmospheric concentrations are produced as a result of a complex series of reactions in the atmosphere, which involve emissions both of nitrogen oxides and of certain reactive hydrocarbons. These emissions are produced from a range of sources, but the most important of these in both North America and western Europe is undoubtedly motor vehicles. The reactions leading to ozone formation are favoured by high temperatures and light intensities, and thus it is characteristically a pollutant of hot summer days. A key feature of such ozone episodes is that high concentrations of the pollutant are not restricted to urban or industrial areas, where nitrogen oxides and hydrocarbons are emitted; instead, high concentrations of ozone may be found over large agricultural regions, and even in remote rural areas (UK PORG, 1993). Indeed, ozone concentrations are often lower in cities than in the surrounding rural areas. It is this wide distribution of high ozone concentrations in rural, as well as urban, areas which is the key reason for its importance as an air pollutant affecting agriculture. Although impacts of ozone on agriculture in North America and western Europe have received considerable attention, there has been little recognition of its potential impacts in the developing countries of Asia, Africa, and South and Central America. Significant impacts of ozone in such countries, where the need to increase food production to meet the requirements of growing populations, and earn foreign exchange, is often vital, could be of much greater economic or social importance than in those parts of the world currently experiencing agricultural

OZONE IMPACTS ON AGRICULTURE

33

surpluses. Emissions of major air pollutants are growing rapidly in many of these countries, with industrialization, urbanization and the growth of transport, while the high temperatures and high solar radiation typical of many of these countries are favourable for the production of high concentrations of ozone. This review aims to provide a critical evaluation of current knowledge of ozone impacts on agriculture in these regions of the world, and to assess the current and future significance of the problem. The first section briefly describes the impacts of ozone on crop physiology and yield, and identifies thresholds for significant impacts on sensitive crops derived from studies in North America and western Europe. These are then compared with the limited data on rural ozone concentrations in other parts of the world to assess the potential impacts of ozone. The available evidence of ozone impacts in the field in developing countries is then described, and the sensitivity of local crops considered. Finally, the significance of the predicted trends of increased emissions of ozone precursors is evaluated.

11.

OZONE IMPACTS ON AGRICULTURAL CROPS

Ozone may have impacts at different levels of organization, from the cellular to individual organs and plants, and to plant communities and ecosystems (Ashmore, 1991). After passing through the stomatal pore, ozone can react with organic molecules in the intercellular space, or with components of the extracellular fluid, leading to the formation of secondary oxidants which can react with the cell membrane. Such reactions can be prevented or reduced by antioxidants, such as ascorbate and polyamines. High concentrations of ozone can cause cells to collapse. leading to visible foliar injury, and effects on the plasma membrane can cause changes in membrane functions which can reduce photosynthetic processes in the chloroplasts. Reduction in COz fixation is typically found in leaves exposed to ozone over longer periods of time (Lenherr et al., 1988). Stimulated dark respiration often also occurs, probably due to increased respiration associated with maintenance and repair (Amthor and Cumming, 1988). The reduced C02 assimilation and increased respiratory C 0 2 loss leads to an overall reduction of assimilate production and export from the source leaves. In leaves of crop species exposed to ozone over long periods, the onset of senescence is typically accelerated (Grandjean and Fuhrer, 1989), and the period with positive net assimilation of CO? is diminished. The reduced overall production of assimilates and altered carbon allocation patterns result in reduced grain or seed yield. Ozone is rarely the only stress factor for crops, and its impact may be modified by a range of other factors. Soil water stress and atmospheric vapour pressure deficit can cause reductions in stomatal conductance and hence in ozone uptake, which may lead to reduced ozone impacts on yield (Fangmeier et nl., 1993). The chemical. physiological and morphological changes to leaves caused by ozone can also alter plant sensitivity to other stresses. There is evidence of such effects for tolerance of cold stress, attack by herbivorous insects and attack by fungal

34

M. R. ASHMORE and F. M. MARSHALL

pathogens. In the case of insect and fungal attack, these effects can be induced by relatively low ozone exposures; for example, ozone at ambient concentrations in south-east England has been shown to increase the performance of insect herbivores on field bean (Ashmore et af., 1987), and increased infestation of fungal pathogens on wheat was observed after one month’s exposure to a low concentration of ozone (von Tiedemann et al., 1991). It is well-established that there are differences between species in their sensitivity to ozone. However, many of the lists of sensitive species are based on visible injury induced by acute ozone exposures; although these are relevant to instances of visible injury in the field, they may not be related to relative sensitivity based on the effects on growth or physiology of longer-term exposures. It is not currently possible to provide comprehensive lists of relative sensitivity of species to these longer-term exposures. Furthermore, the cultivars of widely distributed crops, such as wheat, grown in tropical and subtropical regions may differ substantially in their growth pattern and physiology, and hence their response to air pollutants, from cultivars of more temperate regions.

A. EXPOSURE-RESPONSE STUDIES

The most important source of information on the impacts of ozone on crop yield comes from studies using field chambers in which crops have been exposed to a range of ozone concentrations, and exposure-response relationships for crop yield have been established. Qpically, open-top chambers are placed over field plots of soil-grown plants and supplied with filtered air, unfiltered air, or unfiltered air with ozone added. Such chambers provide climatic conditions that are similar, but not identical, to those outside (Colls et al., 1993). Some reservations about extrapolation to field conditions remain, and recent data (Pleijel et al., 1994) suggest that, due to forced turbulence, the ozone flux in such chambers is normally higher than that outside. This would lead to a tendency to overestimate the adverse effects of a given ozone concentration. Exposure-response data may also be derived from closed chamber studies in glasshouses or controlled environment facilities, although more care is needed in this case in interpreting the significance for field conditions. Complex chamber systems may not always be appropriate or practicable methods of assessing impacts of ozone on crops in developing countries. A useful alternative for ozone is the use of chemical ozone protectants, such as ethylenediurea (EDU), since this avoids the need for experimental enclosures or a power supply (Sanders et al., 1993). Plants treated with EDU can be used as a control and compared with untreated plants, thus providing an estimate of ozone effects. However, because the extent of plant protection from ozone provided is uncertain, these experiments may underestimate the impact of ozone on crops. The first major programme to determine exposure-response relationships for crops was the US National Crop Loss Assessment Network (NCLAN), which was

OZONE IMPACTS ON AGRICULTURE

35

established in the late 1970s, with five experimental sites chosen to reflect the variation in climatic conditions and cropping systems across the country (Heck et al., 1988). Experimental studies involved the use of a standard design of open-top chamber, with a range of ozone concentrations being used in each experiment to generate dose-response relationships between ozone exposure (expressed as the seasonal mean ozone concentration for the seven hours between 9.00 and 16.00) and crop yield. This period was used as it is often the time of day at which the highest ozone concentrations are found. A total of 10 crops were examined (corn, soybean, wheat, hay, tobacco, sorghum, cotton, peanuts, barley and dry beans), representing altogether 85% of the US acreage. Soybean and cotton proved to be among the most sensitive crops to ozone, and barley the least. The programme was designed to allow an estimate to be made of the national impact of air pollution on crop yield. In addition, the dose-response data were integrated with economic models to estimate the value of the loss, and the financial benefits of measures to reduce pollution. The estimate of yield loss nationally requires two other sets of geographical information; data on ozone concentrations nationwide and data on the distribution of the key crops studied in the programme. The results of the study nationally were that a decrease in ozone concentrations of 40% would provide a net annual economic benefit of $3000 million, or about 2.8% of national production (Adams et al., 1988). However, the percentage losses were much higher in particular regions and for specific crops. For example, another study has estimated yield losses due to ozone in California to be 19% for cotton, 23% for dry beans and 24% for onions (Olszyk et ul., 1988). The exposure-response data derived in the NCLAN studies also allow yield losses for different crops to be estimated for a given ozone exposure. For a seasonal mean concentration of 50 ppb, estimated yield reductions for soybean, cotton and forage exceed lo%, whereas that for winter wheat is slightly under 10%; in contrast, at this ozone exposure, estimated yield losses for less-sensitive crops, such as sorghum and rice, were below 5% (Adams et ul., 1988). During the 1980s, an effort was made by the European Commission to develop a similar coordinated assessment of air pollutant impacts on agriculture (Jiiger et ul., 1993). This European Crop Loss Assessment Network had more sites than its US counterpart, but because it was collaborative it had less standardization of chamber design and experimental protocols. A smaller number of crops was studied, with work focusing on wheat and beans; in addition, some studies were conducted on oats and barley. The results showed significant effects of ambient air pollution on the yield of beans and wheat at several locations, although oats and barley appeared to be less sensitive. Unlike NCLAN, the European experimental programme was not specifically designed to estimate regional crop losses through integration with pollution and agricultural datasets. Nevertheless, the data have been employed in a form of agricultural risk assessment, albeit in a rather different way. This is through the establishment of new air quality standards, also termed ‘critical levels’, defined as the air pollutant concentration above which significant adverse effects can occur.

36

M. R. ASHMORE and E M. MARSHALL

The critical level for ozone derived from the European data uses a different method of expressing long-term ozone exposure - the accumulated exposure above a threshold concentration of 40 ppb (AOT40), during daylight hours (Ashmore, 1993; Fuhrer, 1994). Using such an index, linear relationships with crop yield are commonly found. For crop yield losses of 5% and lo%, the AOT40 value has been calculated, based on data for wheat, to be 3000 ppb.h or 6000 ppb.h. respectively (Fuhrer, 1996). The 7 h seasonal mean concentration of 50ppb over an 80 day growing season would correspond to an AOT40 value of 5600 ppb.h, sufficient to cause a 10% yield reduction in wheat. However, it is likely that other European crops, such as Phaseolus vulgaris, for which adequate data are not available to define a critical level, may be more sensitive to ozone (Fuhrer, 1994). No attempts have been made to estimate yield losses due to ozone across Europe. However, for the Netherlands, van der Eerden er czl. ( I 988) estimated that the total yield loss due to all air pollutants was 5%, with 3.4% being due to ozone alone. The impacts of ozone were estimated to be greatest on legumes, potatoes, fodder crops, vegetables and cut flowers. However, these estimates were based on earlier Canadian exposure-response data, rather than the results of the European programmes. Visible foliar injury is a common response to exposure to episodes of high pollutant concentrations, and may adversely affect the value of crop, as well as providing a route for secondary infection. Collaborative European studies of the impact of ozone on visible injury have been conducted using EDU, which provides some protection against ozone injury. The results of the European exercise show a clear north-south gradient, with visible injury being more likely in the warmer areas of southern and central Europe; they also show that there is a risk of visible injury to sensitive crops throughout Europe, except in northern Britain and Scandinavia (Benton et al., 1996).

111. RURAL OZONE LEVELS IN DEVELOPING COUNTRIES The results of the North American and western European studies provide indications of the concentrations of ozone over a crop growing season which may significantly reduce yield. These suggest that yield reductions of 10% or more might be found in sensitive crops when the seasonal mean concentration in the middle of the day exceeds 50 ppb. Extrapolation of these studies to field conditions in the tropics can only be made with caution, but comparison of measured rural concentrations in developing countries can indicate the potential for adverse effects on crop species. However, whereas urban air pollution in cities such as Mexico City, Delhi and Beijing has received considerable attention, and international urban monitoring networks have been established (WHOLJNEP, 1992), there has been very limited coordinated monitoring of air pollution in rural agricultural areas in Asia, Africa or South America. Thus, at the current time, it is only possible to draw on isolated studies in a small number of countries. Table I summarizes information from studies which provide seasonal mean ozone concentrations in the middle ot

37

OZONE IMPACTS ON AGRICULTURE

TABLE I Ozone concentrations in rural areas in developing countries Concentration

Site ..

Brazil - two rural sites in the middle of the sugar cane area in the state of Sao Paolo Brazil - site in the savannah region of central Brazil Mexico - forest area 25 km southwest of Mexico City at an

-.

Reference -

.

7 h mean over 6 days during the dry season at two sites in the range 45-50 ppb

Kirchhoff et al. (1991)

7 h mean for the month of August 1990 about 50 ppb

Kirchhoff et al. ( 1992)

7 h mean concentration about 75 ppb for both summer and winter

Miller et al. (1994)

elevation of 2970 m Egypt - Western Desert 12 h daytime mean 49 ppb 12 h nighttime mean 44 ppb Summer 6 h mean 75-80 ppb Egypt - rural area SO km north of Cairo Seasonal mean midday South Africa - 25 krn concentration about SO ppb from centre of Johannesburg

Gusten et al. (1996) Farag et al. ( 1993)

Stevens ( I 987)

the day, based on continuous monitoring with techniques which measure ozone specifically. One country for which rural ozone data do exist is Egypt, where agricultural production is concentrated in a small area in the Nile valley and delta, which also has high population densities, considerable industrial activity and high traffic densities in some areas. One of the most detailed studies at a rural site in Egypt was carried out by Farag et al. (1993), who made continuous measurements of ozone over the course of one year. The site chosen was about 55 km north of Cairo in an agricultural area. Concentrations reached a maximum between 12.00 and 18.00, and in spring and autumn mean ozone concentrations in this time period were only 30-35 ppb. However, during the summer months, the average concentration was over 75 ppb in this period, with occasional values above 100 ppb. Although these concentrations were lower than those in the city centre and an industrial area of Cairo, they nevertheless indicate the potential for very significant adverse effects on local crops, since substantial impacts of ozone at seasonal mean concentrations of 80 ppb have been shown on a range of American and European crops. The authors suggested that the photolysis of locally applied pesticides could contribute to the high concentrations, but it seems more likely that emissions from Cairo are the major factor involved. These measurements are consistent with data recorded in the summer of 1991

38

M. R. ASHMORE and F. M . MARSHALL

within Cairo by Gusten et al. (1994). The diurnal variation was similar, and the mean afternoon concentrations in mid-summer were about 85 ppb; however, the urban concentrations on occasion reached 120 ppb. Concentrations have also been measured at a remote site in the Western Desert (Gusten et al., 1996). There, as expected, concentrations were lower, but mean daytime concentrations were still close to 50 ppb. In South Africa, Stevens (1987) reported ozone concentrations in the greater Johannesburg region. Although this was essentially an urban study, two of the stations were outside the city boundaries about 25 km from the centre. Concentrations at these sites were higher than in the city centre, and averaged about 50ppb over the summer months during the midday period. On occasional days, the concentration exceeded 120 ppb. Very large concentrations of most major air pollutants have been recorded in Mexico City, where ozone concentrations have reached 400 ppb, and frequently exceed 150 ppb. In Brazil, high concentrations of ozone are common in Sao Paolo and Cubatao, with values frequently exceeding 80 ppb, and reaching 200 ppb on occasions. In both countries there are few comparable data on ozone levels in the agricultural areas surrounding the large cities. However, the limited information which is available does indicate the potential for ozone impacts on vegetation. Ozone concentrations have been monitored in a mountain area close to Mexico City, where damage to forests has been reported (Miller et al., 1994), and compared to those in forests around Los Angeles where extensive ozone damage has been documented. The concentrations are lower at the Mexican site in summer, but higher in winter, and the Mexican site experiences 7 h mean concentrations in the middle of the day averaging about 75 ppb, throughout the year. A number of studies have been carried out in remote areas of South America as part of studies of global background tropospheric ozone. In general, these studies reveal ozone concentrations of 15-30 ppb, below those of any concern in terms of impacts on vegetation. However, there is some evidence of increased ozone concentrations during the dry season, when biomass burning is occurring in the region. Thus, Kirchoff et al. (1992) measured ground-level ozone at sites in an area of central Brazil dominated by cerrado vegetation during the dry season; the sites were chosen to avoid the influence of any urban sources of ozone precursors. Concentrations of ozone, expressed as monthly 7 h mean concentrations, reached 50 ppb during this period. Kirchoff et al. (1991) also demonstrated that burning of sugar cane fields could contribute to the formation of significant ozone concentrations; this source has increased considerably since the introduction of the use of alcohol as a substitute for gasoline in the early 1980s. Measurements made at two rural sites in the state of Sao Paolo, during the dry season of 1990, showed mean ozone concentrations in the middle of the day reaching about 50 ppb. These values were similar to those recorded simultaneously at an urban site, indicating that sugar cane burning can contribute to ozone formation at this time of year to a similar extent to urban transport and industry.

OZONE IMPACTS ON AGRICULTURE

39

Data from the field studies in Pakistan described in Section IV demonstrate the presence of elevated ozone concentrations in the middle of the day at a site in the suburbs of Lahore, with mean midday concentrations reaching 60 ppb in some summer months (Maggs ef al., 1995). However, these measurements were made using a chemical method which may respond to other atmospheric oxidants, such as nitrogen dioxide, and thus they must be interpreted with some caution. Other data from urban sites in India, using a range of different analytical methods, have demonstrated 7 h mean ozone concentrations in summer months which exceed 40 ppb, and in some cases approach 60 ppb (Thimmiah, 1996). The evidence from the Indian subcontinent generally suggests the potential for generation of ozone in concentrations high enough to damage sensitive crops in and around large cities, but there is a lack of continuous monitoring using ozone-specific methods in rural areas. In summary, the limited data available demonstrate clearly that seasonal mean ozone concentrations at a number of sites fall in the range 50-80ppb, at which adverse effects on the yield of sensitive crops might be expected. At several of these sites, pollutant emissions from neighbouring cities are likely to be primarily responsible, but seasonal biomass burning may be a further additional source in certain areas and seasons. There is a clear need both to collate additional datasets which are not accessible through journal publications, and to increase the extent of ozone measurement in agricultural areas likely to be affected by urban or biomass emissions.

IV. DIRECT EVIDENCE OF ADVERSE EFFECTS ON CROPS The data summarized in Table I thus raise the possibility of adverse impacts of ozone on agricultural crops in developing countries. Direct experimental evidence to support this assertion comes from two major sources: field chamber studies in which air has been filtered to remove ambient pollutants, and field trials in which plants have had ozone protectant chemicals applied. Table I1 summarizes some of the key studies which have been carried out.

A.

STUDIES WITH FIELD CHAMBERS

The use of field chambers which are ventilated with unfiltered air, or with air filtered through activated charcoal to remove air pollutants, has proved an effective tool in identifying adverse effects of the ambient air pollutant mix in Europe. However, few such experiments have so far been carried out with field chambers in developing countries. The most important series of experiments of this nature is that using local cultivars of rice and wheat at a site on the outskirts of Lahore, in an area where these crops are being grown (Maggs et al., 1995a,b; Wahid et al., 1995a,b). Two cultivars of each species were grown in pots in the chambers under

TABLE I1 Field experiments on the effects of ozone on crops in developing countries Method

Reference

Response ~

Pakistan Punjab in the viciniw of Lahore

Or~m sativa cv Basmati-385 and IRRI-6

Pakistan Punjab in the vicinity of Lahore

Triticum aestivum cv Pal-81 and cv Chakwal-86

Pakistan Punjab; 3 locations in the vicinity of Lahore Indian Punjab

Glycine

cv NARC 1

) 7 2 ~

Open-top chambers with charcoal-filtered air to remove 0, and other pollutants Open-top chambers with charcoal-filtered air to remove O3 and other pollutants Application of EDU

Solanum tuberosurn cv. Kufri jyoti

Dusting with activated charcoal or addition of EDU

Abbis 35 km south of Alexandria, Egypt

Raphanus sativus and Brassica rapa

Application of EDU

Montecillos, Mexico; a rural site in the Valley of Mexico

Phaseolus vulgaris cv Canario 107 and Pinto 111

Application of EDU

42% yield loss in unfiltered air for Basmati-385; 37% loss for LRRI-6 46.7% yield loss in unfiltered air for Pak-81; 34.8% for Chakwal-86 49% reduction in seed wt. in untreated plants at the rural site Treated plants did not develop visible injury, whereas untreated plants did Root and shoot dry weight decreased by 30 and 17% in radish, and 17 and 11% in turnip, in untreated plants 4.5% yield reduction in untreated plants of Canario 107; 40.7% yield reduction in untreated plants of Pinto III

~

~~

~

Wahid et al. (1995a)

Wahid et al. (1995b)

Wahid, A. and Shamsi, S. R. A. (pers. comm.) Bambawale (1986)

Hassan et al. (1995)

Laguette Rey et al. (1986)

OZONE IMPACTS ON AGRICULTURE

41

local cultivation conditions in two successive growing seasons (NovemberApril/May for wheat, and May/June-OctoberlNovember for rice). Plants were also grown outside the chambers, to demonstrate that there was relatively little effect of the chamber enclosure on crop growth and yield. All four experiments showed a large and significant effect of filtration on the yield of both species, with yield in unfiltered air being reduced by 34% and 45% for the two wheat cultivars (Pak-81 and Chaknwal-86), and by 37% and 46% for the two rice cultivars (Basmati-385 and IRRI-6), averaged over the two years. The most important yield component affected by filtration was the number of ears, or number of panicles, per plant, and in all cases, leaf sensescence was accelerated in unfiltered air. In contrast, effects of filtration on 1000 grain weight were relatively small. The concentrations of sulphur dioxide at this site were negligible, but there were significant concentrations of nitrogen dioxide and ozone. Mean nitrogen dioxide concentrations were typically about 25 ppb, except during the monsoon season, when they were much lower. Concentrations of ozone, determined using a chemical method which determines total oxidant levels, varied between 40 and 60-70 ppb, as a 6 h mean concentration, on individual days, except during a heavy monsoon period and cool winter periods. Concentrations of ozone were generally higher in the rice season than the wheat season. The recorded concentrations of nitrogen dioxide are lower than those normally found to significantly reduce crop growth and yield, and this was confirmed by laboratory studies of these particular cultivars (Maggs, 1996). Thus it is likely that the large yield reductions observed in this experiment were primarily due to ozone. The size of the yield reductions found is larger than has been reported at similar concentrations in controlled fumigation studies with wheat and rice (e.g. Kats er al., 1985; Kohut et ui., 1987; Pleijel et nl., 1991; Kobayashi, 1993). This may be due to differences in cultivar sensitivity or climate. The results of these experiments imply that ozone may be having a substantial impact on rice and wheat yields in the Pakistan Punjab, but until the work is repeated at other locations, it is impossible to be sure that the results do not represent the impact of an unusual air pollutant mix at the particular experimental site.

B. STUDIES WITH OZONE PROTECTANT CHEMICALS

The first experiment in a developing country to use the ozone protectant N-[2-(2-oxo- I -imidazolidinyl)ethyl]-N’-phenylurea(EDU) to assess the impacts of ambient ozone in a rural location was that of Bambawale (1986), who tested the hypothesis that leaf spot on Solanurn tuberosurn was caused by ozone. The work was carried out near Jalanghar, in northern India. and showed that application of both EDU and activated charcoal dust to screens above the plants reduced the prevalence of the symptom. Work reported in the same year by Laguette Rey et al.

42

M. R. ASHMORE and F. M. MARSHALL

( 1986) in Montecillos in Mexico demonstrated that application of EDU increased the yield of one cultivar of Phaseolus vulgaris by 41%, although the effect on a second variety was only 5%. This work was carried out in an area close to Mexico City where characteristic visible foliar symptoms of ozone damage have been found in a number of crops. Given the diagnostic value of this work, it is surprising that relatively few further studies have been carried out over the subsequent decade. The only recent published study with EDU in developing countries appears to be that of Hassan et al. (1 993, who grew Egyptian cultivars of radish and turnip at two sites: one in the suburbs of Alexandria, and one in a village (Abbis) in the Nile delta, 35 km south-east of Alexandria. The harvested dry weights of both species were significantly reduced at Abbis, by 30% for radish and by 17% for turnip, in plants not treated with EDU; at Alexandria, the dry weight of turnip was not significantly altered by EDU treatment, whereas that of radish was reduced by 24% in untreated plants. EDU also effectively reduced visible symptoms of ozone injury which appeared on radish at both sites, and on turnip only at the village site. The fact that the value of EDU protection was greater at the remote rural site than at the suburban site is consistent with measurements of ozone concentrations at the two sites, which showed higher values at the more rural site. This result is also consistent with studies in the US and in Europe, and can be explained by the removal of ozone by reaction with nitric oxide (NO) at more urban sites. The 6 h mean oxidant levels recorded during the experiment, in February and March, were 55 ppb in Alexandria and 67 ppb in the village site. It is probable that higher concentrations, with the potential for larger effects on yield, would be found in the summer months. Recently, A. Wahid and S . R. A. Shamsi (personal communication) have completed a similar experiment, with a Palustani cultivar of Glycine mar, in and around Lahore. A key element of the experiment was a comparison of the protective effect of EDU at the site on the outskirts of Lahore used in the chamber experiments, and at a rural site about 35 km east of the city. The seed weight per plant was 32% lower in the untreated plants, compared with the EDU-treated plants, at the suburban site, and 49% lower at the rural site. As in the Egyptian experiment, the larger difference in yield between the EDU-treated and control plants at the more rural site was associated with higher atmospheric oxidant levels. The studies with EDU in Mexico, Pakistan and Egypt clearly demonstrate the potential for ozone to cause large impacts on yield at rural sites close to major cities. Without further research it is impossible to be certain to what extent this effect would extend into more remote rural areas. However, given the large size of the effects observed in these experiments, and the fact that EDU may not prevent all adverse effects of ozone on crop yield (Hassan er al., 1995). this limited body of evidence clearly indicates that the impacts of ozone may be a very significant problem in the field.

OZONE IMPACTS ON AGRICULTURE

43

V. RESPONSES TO OZONE OF TROPICAL CROPS AND CULTIVARS A. EXPERIMENTAL STUDIES

Studies using chambers in glasshouses or controlled environment rooms cannot replicate field conditions, but they may indicate the sensitivity of crops to ozone, as well as determining the mechanisms leading to the observed effects of ozone. Again, limited experimental data are available on the response of tropical crops or cultivars to ozone, and many of the experiments which have been carried out have used seasonal daylight mean concentrations well above the maximum of 80 ppb so far recorded at a rural site in a developing country (Table I). More experiments exist on the responses of crops such as wheat and rice, but these have used North American, European or Japanese cultivars, the responses of which may be quite different from the local cultivars. One important series of laboratory fumigation experiments which has been recently completed is that of Maggs (1996), who studied the response to ozone of the Pakistani cultivars of wheat and rice which were used in the filtration studies with open-top chambers on the outskirts of Lahore (Wahid et al., 1995a,b). The results are summarized in Table 111. The experiment with wheat involved exposing both of the cultivars of wheat used in the field chamber experiments to ozone for 8 h per day, on 5 days per week, over a period of 11 weeks. The 8 h mean concentration on the fumigation days was 65 ppb, although when expressed as a mean over the entire 1 I weeks it was 46 ppb. The plants were also exposed to 30 ppb nitrogen dioxide, alone or in combination with ozone; however, there was no significant effect of this concentration of nitrogen dioxide on plant growth or yield. In contrast, ozone reduced the yield per plant by 48% for Pak-81, and by 31% for Chak-86. Both cultivars showed a similar reduction, of about 30%, in 1000 grain weight, and the reason for the larger yield effect in Pak-81 was additional significant ozone effects on grain number per plant and percentage sterility. This experiment strongly suggests that the impacts of ambient air at the study site in Lahore were due to ozone, rather than nitrogen dioxide, and also demonstrates the sensitivity of the cultivars to ozone. However, the nature of the yield components most affected by ozone in these laboratory experiments differed from those in the field study. A similar experiment to examine the effects of ozone and nitrogen dioxide, alone or in combination, was carried out by Maggs (1996), with two cultivars of rice. However, this experiment was only of 40 days duration, and used lower concentrations - the 8 h mean ozone concentration on fumigation days was 47 ppb, whereas the 24 h mean nitrogen dioxide concentration was 17 ppb. As for wheat, there was no significant effect of nitrogen dioxide, alone or in combination with ozone, but ozone alone did significantly reduce vegetative biomass, by 19% in the case of Basmati-385 and by 18% in the case of IRRI-6. In this experiment, as in the

TABLE 111 Fumigation studies with ozone on crops or cultivars grown in developing countries Crop

Method

_ _ _ _ _ _ _ ~ ~ . _ _ _ _ _ ~

O3 exposure ~

Cicer arietinum

Fumigation in closed polythene chambers

80 ppb O3 for 2 hlday for 20 days

vicia faba

Fumigation in closed polythene chambers

80 ppb O3 for 1.5 hlday for 60 days

Triticum aestivum

Fumigation in closed glasshouse chambers

65 ppb O3 for 8 h/day for 5 days/week for 11 wks

Oryza sativa

Fumigation in closed glasshouse chambers

67 ppb O3 for 8 hlday for 40 days

Oryza sativa

Closed glasshouse chambers Fumigation in greenhouse domes

54 ppb O3 for 8 Wday for 133 days 70 ppb O3 for 7 Wday for 6 weeks

13 leguminous species and I fibre crop

~

_

Response _

_

Grain wt. reduced by 77% Reduced nodule size and number Chlorosis within 15 days Vegetative dry wt. reduced by 50% Reduced chlorophyll and protein content 48% reduction in grain wt. in cv Pak-81 31% reduction in grain wt. in cv Chakwal-86 19% reduction in vegetative dry wt. in cv Basmati-385 18% reduction in vegetative dry wt. in cv IRFU-6 57% reduction in grain wt. in cv. DRRI-6 Vegetative dry wt. reduced by more than 20% in Medicago sativa, Vigna radiata, Vigna mungo and Hibiscus cannabinus

~~~~

_

Reference

~

Singh and Rao (1982)

Agrawal et al. (1985)

Maggs (1996)

Maggs (1996)

Maggs (1996) Kasana (1988)

OZONE LMPACTS ON AGRICULTURE

45

earlier experiment with wheat, a major effect of ozone was to accelerate leaf sensescence, a phenomenon observed in many other studies of ozone on cereals, including the field chamber filtration study in Pakistan. A final, longer experiment, over 165 days, examined the effect of ozone alone on rice cv. IRRI-6. The mean 8 h concentration of ozone on fumigation days was 54 ppb (43 ppb over the entire experiment). This treatment caused a very large reduction, of 5796, in the total grain weight per plant. This was the result of a combination of reduced numbers of spikelets per plant, reduced panicle number per plant, increased spikelet sterility, and reduced 1000 grain weight. As in the other experiment, a major effect of ozone during the development of the crop was to accelerate leaf sensescence. The results of these experiments clearly demonstrate the sensitivity of the cultivars used to ozone, and indicate the potential for substantial yield reductions in the field. However, Maggs (1996) points out that the grain yield per plant achieved using these cultivars in the fumigation experiments was much lower than that in the field experiments in Pakistan, and thus extrapolation to field conditions must be made with caution. Table 111 also summarizes three studies of Indian crops or cultivars which have used realistic ozone exposures, in the range 50-80 ppb. Singh and Rao (1982) used a concentration of 80 ppb, for 2 h per day, to study ozone effects on gram (Cicer ariptinuni) plants, which were 90 days old. Within 15 days, chlorotic spots had appeared on the upper leaf surface, and both leaf number and root nodule number were significantly reduced, and plants harvested after 30 days showed large and significant reductions in both pod number and mean pod weight. Agrawal et ul. (1985) used a similar exposure regime in a longer-term study of Vicia faha. After 30 days of exposure, numbers of leaves and root nodules, protein and chlorophyll contents, and foliar nitrogen and phosphorus contents were all significantly reduced in the ozone treated plants. Whereas these two experiments were carried out in India, Kasana (1988) fumigated a range of Indian crops in an outdoor fumigation facility in the UK. The work focused on leguminous crops in view of their established sensitivity to ozone, and their importance as sources of both food and fodder in many tropical and subtropical countries. Exposure to 70ppb, for 7 h per day, for 6 weeks had relatively little effect on the vegetative dry weight of Cicer nrietinum, Cnjmus Cajun, Lens culinuris and Vigna unguicuhta. However, other tested species showed growth reductions of more than 20%. These were Medicagn sativa, Vigna rndiatu, Vigna mungo, in which the growth reduction was 60% and the fibre crop Hibiscus cannubinus.

B. FACTORS INFLUENCING OZONE SENSITIVITY IN THE FIELD

The experiments described in the previous sections are very limited, and also involve the growth of plants in pots, supplied with adequate water, and protected

46

M. R. ASHMORE and F. M. MARSHALL

from pests and diseases. In assessing the real influence of ozone on cropping systems under field conditions in tropical and subtropical areas, a number of other factors need to be taken into account, in addition to the actual ozone exposures at different locations. First, the time of year in which the crop is grown is of considerable significance. Many air pollutants have a typical seasonal cycle. In general terms, ozone levels tend to be higher in the summer, when conditions are more conducive to its formation. However, this will not apply in seasons with heavy rainfall, and in dry seasons, biomass burning may be an additional contributing factor. Furthermore, in the tropics, climatic conditions favourable to ozone production may continue almost throughout the year. The interaction of these factors with local cropping patterns needs further assessment. Secondly, the nature of the cropping systems may influence its response to ozone. The most obvious factor will be irrigation - in general, well-watered plants have a higher pollutant uptake and thus show greater sensitivity than rain-fed crops. However, there are a number of other factors, such as atmospheric humidity, temperature, salinity and fertilizer levels which may influence the responses of crops to air pollutants. A particularly important factor may be the known impacts of relatively low concentrations of air pollutants in influencing the performance of insect pests and plant pathogens (Bell et al., 1993). Finally, the choice of cultivar will also be important, since there is known to be a wide variation in sensitivity between different cultivars in pollution sensitivity. The issues of whether there are systematic differences between, for example, high-yielding cultivars and more traditional cultivars, and between cultivars bred in areas with high or low air pollution levels, are of considerable practical significance. The introduction of new, more tolerant cultivars, has proved effective in certain cases in the US in reducing the impacts of ozone; if the pollutant is indeed reducing crop yields in certain areas of the developing world, the ability to identify alternative cultivars with greater tolerance could offer the opportunity to reduce this yield loss more rapidly than might be feasible by addressing pollutant emissions.

VI. FUTURE CONCENTRATIONS AND IMPACTS OF OZONE There is now good observational data to suggest that the global background tropospheric ozone concentration is increasing as a result of human activities (Penkett, 1988; Hough and Dement, 1990). The production of ozone in the background troposphere is limited by emissions of nitrogen oxides, and should increase as these emissions increase. Thus, evidence of increased emissions of nitrogen oxides, and increases in nitrate concentrations in ice cores in remote areas, such as Greenland, supports the empirical evidence of a rising trend of background tropospheric ozone concentrations, linked to increased global emissions of nitrogen oxides.

OZONE IMPACTS ON AGRICULTURE

47

Chameides et al. (1994) used a global model to examine in more detail the increases in ozone concentrations as a result of increased nitrogen oxide emissions, focusing on three ‘continental-scale metro-agro-plexes’. These areas, comprising eastern North America, Europe, and east China and Japan, are regions where currently high energy consumption, high population densities and intensive agricultural production coincide - the authors estimate they are responsible for 75% of global energy and fertilizer production, and 60% of global food production and export. The predicted rise in nitrogen oxide emissions by 2025 is particularly marked in East Asia. The increases in nitrogen oxide emissions predicted by Chameides et al. (1994) were then used to estimate increases in global ozone levels, which were then linked to data on ozone impacts on cereals. As discussed above, these suggest that the threshold for significant yield reductions in sensitive cereals, such as wheat, is 50ppb, whereas that for less sensitive cereals, such as rice, is 70ppb - both concentrations being expressed as 7 h seasonal mean concentrations. Using this model, the increase in nitrogen oxide emissions predicted for 2025 will increase ozone impacts significantly. Nitrogen oxide emissions are predicted to increase globally from 110 kT day-’ in the mid 1980s to 150-180 kT day-’ in 2025, depending on the economic scenario used. This change is predicted by Chameides et al. (1994) to increase the percentage of the world cereal crop exposed to ozone levels above the threshold of 50-70 ppb from 9-35% to 30-75%. Much of the increase in emissions predicted by Chameides et al. (1994) will occur outside North America and western Europe. Although the use of fossil fuels in power generation, transport and industry will be an important cause of the increased emissions, Chameides et al. ( 1994) also calculate that increased fertilizer use will be another important contributor to increased NO, emissions. Although the details may vary, other sources are in broad agreement with a scenario of significantly increased emissions of nitrogen oxides by 2020, concentrated particularly in Asia, resulting from nitrogenous fertilizer use, as well as fossil fuel usage, and that this will result in increased ground-level ozone concentrations (Galloway, 1989; Galloway el al., 1994, 1995; Houghton et al., 1996). Galloway (1995) emphasizes the importance of the increases in Asia, in particular, where the increases in fossil fuel and fertilizer use needed, under current scenario assumptions, to sustain 50% of the world’s population will be very large in absolute terms.

VII. CONCLUSIONS It is clear from this review that the amount of information available on rural ozone levels, and on the responses of local crops to these levels, is so small that no coherent analysis of the impacts of the pollutant on agriculture in developing countries is possible. Nevertheless, the evidence which does exist indicates that the effect of ozone in many areas of such countries could already be substantial.

48

M. R. ASHMORE and F. M. MARSHALL

Clearly, more research is urgently needed to assess the potential impacts of ozone more thoroughly. Enough information is available to identify the crops and regions globally where the problem might be of greatest concern. These are likely to be areas of the wet tropics, or the dry tropics where agriculture is based around irrigation, where emission densities of nitrogen oxides are relatively high. Key areas may thus be south and south-east Asia, parts of central and south America, and parts of north Africa. The evidence summarized in this paper indicates that ozone impacts on agriculture are no longer a problem only in North America and western Europe. It is clear that, in the future, this is likely to become more of a global issue, for two reasons. First, in many regions of the planet, local urban or industrial emissions of nitrogen oxides are likely to increase substantially over the next 30 years, producing polluted air masses bringing ozone into the surrounding rural areas. Secondly, the steady increase in global emissions of nitrogen oxides over the same period means that an increase in global background tropospheric ozone levels is likely. Where large local or regional increases are superimposed on the steadily increasing global background level, the impact may be considerable. It is thus possible that ozone concentrations are already, or could become, a significant constraint on national or regional agricultural production in a number of countries in which substantial increases in food production are needed to feed growing populations. Much of the attention concerning the issue of air pollution in developing countries is currently focused on the impacts on human health in large cities. However, air pollution impacts on agriculture in and around these cities could have significant economic and social impacts, and thus indirect impacts on public health. The importance of the issue has not yet been recognized by national or international agencies. Although this review has concentrated on ozone, as the most important pollutant on national or regional scales, the impacts of sulphur dioxide, in particular, at a more local scale also need more recognition. Air pollution monitoring has tended to focus on urban areas, and there has been little rural monitoring; this is a particular deficiency in the case of ozone, which may be of considerable significance in rural areas. There is a clear and urgent need to develop collaborative international experimental programmes to assess the current and future significance of ozone impacts on agriculture on a more global basis. The development of air quality guidelines for agriculture, which are related to local cropping patterns and climatic conditions, would also be helpful in guiding policy development in this area.

ACKNOWLEDGEMENTS We acknowledge the contribution of our colleagues Nigel Bell and Eleanor Milne to developing many of the ideas expressed in this paper. Our work on air pollution impacts on agriculture in developing countries has been supported by the Scientific

OZONE lMPACTS ON AGRICULTURE

49

Cooperation Programme of the European Community, and by the Environmental Research Programme of the Department for International Development.

REFERENCES Adams, R. M., Glyer, J. D. and McCarl, B. A. (1988). The NCLAN economic assessment: approach, findings and conclusions. I n “Assessment of Crop Loss from Air Pollutants” (W. W. Heck, 0. C. Taylor and D. T. Tingey, eds) pp. 473-504. Elsevier, New York. Agrawal, M., Nandi, P. K. and Rao, D. N. (1985). Responses of Viciafaha plants to ozone pollution. Indian Journal of Environnzenral Heulth 27, 3 18-329. Amthor, J . S . and Cumming, J. R. (1988). Low levels of ozone increase bean leaf maintenance respiration. Canadian Journal of Botany 66, 724-726. Ashmore, M. R. (1991). Air pollution and agriculture. Outlook on Agriculture 20, 139-144. Ashmore, M. R. (1993). Critical levels and agriculture in Europe. hi “Effects of Air Pollution on Agricultural Crops in Europe” (H. J. Jager, M. H. Unsworth, L. de Temmerman and P. Mathy, eds) pp. 105-129. Air Pollution Research Report 46, Commission of the European Communities, Brussels. Ashmore, M. R., Brown, V., Kristiansen, L. and Shah, D. (1987). Effects of ambient air pollution, water stress and aphid pests on Viciufuha. In “The European Communities Research Project on Open-Top Chambers. Results on Agricultural Crops 1987-1988” (J. Bonte and P. Mathy, eds) pp. 8-25. Commission of the European Communities, Brussels. Bambawale, 0. M. (1986). Evidence of ozone injury to a crop plant in India. Atniuspheric Environment 20, 1501-1503. Bell, J. N. B., McNeill, S., Houlden, G., Brown, V. C. and Mansfield, P. J. (1993). Atmospheric change: effect on plant pests and diseases. Parasifology 106, S11- S24. Benton, J., Fuhrer, J., Cimeno, B. S., Skarby, L., Palmer-Brown, D., Roadknight, C. and Sanders-Mills, G. (1996). The critical level of ozone for visible injury on crops and natural vegetation (IC-Crops). In “Critical levels for Ozone in Europe: Testing and Finalising the Concepts” (L. Karenlanipi and L. Skarby, eds) pp. 44-57. University of Kuopio, Kuopio. Chameides, W. L., Kasibhatia, P. S., Yienger, J. and Levy, H. I1 (1994). Growth of continental-scale metro-agro-plexes, regional ozone pollution, and world food production. Science 264, 74-77. Colls, J. J., Weigel, H. J. and Geissler, P. A. (1993). Microclimate in open-top chambers. In “Effects of Air Pollution on Agricultural Crops in Europe” (H. J. Jager, M. H. Unsworth, L. de Temmernian and P. Mathy, eds) pp. 175-191. Air Pollution Research Report 46, Commission of the European Communities, Brussels. Fangmeier, A., Galaup, S . and Bonte, J. (1993). CEC assessment on drought stress effects. In “Effects of Air Pollution on Agricultural Crops in Europe” (H. J. Jager, M. H. Unsworth, L. de Temmennan and P.Mathy, eds) pp. 4 3 3 4 . Air Pollution Research Report 46, Commission of the European Communities, Brussels. Farag, S. A,, Rizk, H. F. S . , El-Bahnasaway, R. M. and Meleigy, M. I. (1993). The effect of pesticides on surface ozone concentrations. International Journal of Environmental Edurution und Information 12, 2 17- 224. Fuhrer, J. (1994). The critical level for ozone to protect agricultural crops - an assessment of data from European open-top chamber experiments. In “Critical Levels for Ozone; a UN-ECE workshop report” (J. Fuhrer and B. Achermann, eds) pp. 42-57. FAC Report no. 16, Swiss Federal Research Station for Agricultural Chemistry and Environmental Hygiene, Liebefeld-Bern.

50

M. R. ASHMORE and E M. MARSHALL

Fuhrer, J. (1996). The critical level for effects of ozone on crops, and the transfer to mapping. In “Critical Levels for Ozone in Europe: Testing and Finalising the Concepts” (L. Karenlampi and L. Skarby, eds) pp. 2 7 4 3 . University of Kuopio, Kuopio. Galloway, J. N. (1989). Atmospheric acidification: projections for the future. Ambio 18, 161-1 67. Galloway, J. N. (1995). Acid deposition: perspectives in time and space. Wafer;Air and Soil Pollution 85, 15-24. Galloway J. N., Levy, H. I1 and Kashibhatia, P. S. (1994). Year 2020: consequences of population growth and development on deposition of oxidised nitrogen. Ambio 23, 120-1 23. Galloway, J. N., Schlesinger, W. H., Levy, H. 11, Michaels, A. and Schnoor, J. L. ( 1995). Nitrogen fixation: anthropogenic enhancement-environmental response. Global Biogeochemical Cycles 9, 235-252. Grandjean, A. and Fuhrer, J. (1989). Growth and leaf senescence in spring wheat (Triticum aestivwn) grown at different ozone concentrations in open-top field chambers. Physiologia Plantarum 77, 389-394. Gusten, H., Heinrich, G., Weppner, J., Abdel-Aal, M. M., Abdel-Hay, F. A., Ramadan, A. B., Tawfik, F. S., Ahmed, D. M., Hassan, G. K. Y.,Cvitas, T., Jeftic, J. and Klasine, L. (1994). Ozone formation in the greater Cairo area. Science of rhe Total Environment 155, 285-295. Gusten, H. E., Heinrich, G.,Monnich, E., Sprung, D., Weppner, J., Ramadan, A. B., Ezz El-Din, M. R. M., Ahmed, D. M. and Hassan, G. K. Y. (1996). On-line measurements of ozone surface fluxes: Part 11. Surface-level ozone fluxes onto the Sahara desert. Atmospheric Environment 30, 91 1-918. Hassan, I. A., Ashmore, M. R. and Bell, J. N. B. (1995). Effect of ozone on radish and turnip under Egyptian field conditions. Environmental Pollution 89, 107-1 14. Heck, W. W., Taylor, 0. C. and Tingey, D. T. (eds) (1988). “Assessment of Crop Loss from Air Pollutants”. Elsevier Applied Science, London. Hough, A. M. and Derwent, R. G. (1990). Changes in the global concentration of tropospheric ozone due to human activities. Nature 344, 645-650. Houghton, J. T., Meira Filho, L. G., Callander, B. A., Harris, N., Kattenberg, A. and Maskall, K. (1996). “Climate Change 1995. The Science of Climate Change”. Cambridge University Press, Cambridge. Jager, H. J., Unsworth, M., De Temmerman, L. and Mathy, P. (eds) (1993). “Effects of Air Pollution on Agricultural Crops in Europe”. Air Pollution Research Report 46, Commission of the European Communities, Brussels. Kasana, M. S. (1988). High relative sensitivities of some leguminous crops to ozone pollution. Environmental Conservation 356-358. Kats, G., Dawson, P. J., Bytnerowicz, A., Wolf, J. W., Thompson, C. R. and Olszyk, D. (1985). Effects of ozone and sulfur dioxide on growth and yield of rice. Agriculture Ecosystems and Environment 14, 103-1 17. Kirchoff, V. W. J. H., Marinho, E. V. A., Dias, P. L. S., Pereira, E. B., Calheiros, R., Andre, R. and Volpe, C. (1991). Enhancements of CO and ozone from burning in sugar cane fields. Journal of Atmospheric Chemistry 12, 87-102. Kirchoff V. W. J. H., Nakamura, Y., Marinho, E. V. A. and Mariano, M. M. (1992). Excess ozone production in Amazonia from large-scale burnings. Journal ofdtmospheric and Terrestrial Physics 54, 583-588. Kobayashi, K. (1993). Findings of a modelling effort for assessing ozone impact on rice production. In “Air Pollution and Crop Responses in Europe” (H. J. Jager, M. H. Unsworth, L. de Temmerman and P. Mathy, eds) pp. 550-553. Commission of the European Communities, Brussels.

OZONE IMPACTS ON AGRICULTURE

51

Kohut, R. J., Amundsen, R. G., Laurence, J. A., Colavito, L., van Leuken, P. and King, P. (1987). Effects of ozone and sulfur dioxide on the yield of winter wheat. Phytoputhology 77, 71-74. Laguette Rey, H. D., de Bauer, L. I., Shibata, J. K. and Mendoza, N. M (1986). Impact0 de 10s oxidante ambtales en el cultivito de frijol, en Montecillos, estado de Mexico. Centro de Fitopatologia 66, 83-95. Lehnherr, B., Machler, F., Grandjean, A. and Fuhrer, J. (1988). The regulation of photosynthesis in leaves of field-grown spring wheat (Triticum aestivum L., cv Albis) at different levels of ozone in ambient air. Plant Physiologv 88, 1115-1 119. Maggs, R. (1996). “The effects of ozone and nitrogen dioxide on Pakistan wheat (Triricum aestivum L.) and rice (Oryza sariva) cultivars”. PhD thesis, University of London. Maggs, R., Wahid, A., Shamsi, S. R. A. and Ashmore, M. R. (1995). Effects of ambient air pollution on wheat and rice yield in Pakistan. Watel; Air and Soil Pollution 85, I3 11-1 3 16, Miller, P.. de Bauer, L. I., Nolasco, A. Q. and Tejeda, T. H. (1994). Comparisons of ozone exposure characteristics in forested areas near Mexico City and Los Angeles. Atmospheric Environment 28, 141-148. Olszyk, D. M., Thompson, C. R. and Poe, M. P. (1988). Crop loss assessment for California: modelling losses with different ozone standard scenarios. Environmental Pollution 53, 303-3 11. Penkett, S. A. (1988). Indications and causes of ozone increase in the troposphere. In “The Changing Atmosphere” (F. S . Rowland and I. S. A. Isaksen, eds) pp. 91-102. John Wiley, London. Pleijel, H., Skarby, L., Wallin, G. and SelldCn, G. (1991). Yield and grain quality of spring wheat (Triticum aestivum cv. Drabant) exposed to different concentrations of ozone in open-top chambers. Environmental Pollution 69, 15 1-168. Pleijel, H., Wallin, G., Karlsson, P. E., Skiirby, L. and SelldCn, G. (1994). Ozone deposition to an oat crop (Avena sativa L.) grown in open-top chambers and in the ambient air. Atmospheric Environment 28, 1971-1979. Sanders, G . E., Booth, C. E. and Weigel, H. J. (1993). The use of EDU as a protectant against ozone pollution. In “Effects of Air Pollution on Agricultural Crops in Europe” (H. J. Jager, M. H. Unsworth, L. de Temmerman and P. Mathy, eds) pp. 359-369. Air Pollution Research Report 46, Commission of the European Communities, Brussels. Sanders, G. E., Skarby, L., Ashmore, M. R. and Fuhrer, J. (1995). Establishing critical levels for the effects of air pollution on vegetation. Water; Air and Soil Pollution 85, 189-200. Singh, M. and Rao, D. N. (1982). The influence of ozone and sulphur dioxide on Cicer arietinum L. Journal of Indian Boranicul Societ?, 61,51-58. Stevens, C. S. (1987). Ozone formation in the greater Johannesburg region. Atmospheric Environment 21, 523-530. Thimmiah, S. (1996). “Air pollution in India with respect to deleterious impacts on agriculture”. MSc thesis, Imperial College Centre for Environmental Technology, London. UK PORG (1993). “Ozone in the United Kingdom 1993”. United Kingdom Photo-oxidant Review Group, 3rd Report. Department of the Environment, London. van der Eerden, L. J., Tonneijck, A. E. G. and Wijnands, J. H. M. (1988). Crop loss due to air pollution in the Netherlands. Environmental Pollution 53, 365-376. von Tiedemann, A., Weigel, H. J. and Jager, H. J. (1991). Effects of open-top chamber fumigations with ozone on three fungal leaf diseases of wheat and the mycoflora of the phyllosphere. Environmental Pollution 72, 205- 224.

52

M. R. ASHMORE and F. M. MARSHALL

Wahid, A., Maggs, R., Shamsi, S. R. A,, Bell, J. N. B. and Ashmore, M. R. (1995a). Air pollution and its impacts on wheat yield in the Pakistan Punjab. Envimnmentul Pollution 88, 147-154. Wahid, A., Maggs, R.,Shamsi, S. R. A., Bell, J. N. B. and Ashmore, M. R. (1995b). Effects of air pollution on rice yield in the Pakistan Punjab. Environmental Pollution 90, 323-329. WHORJNEP (1992). “Urban Air Pollution in the Megacities of the World”. World Health Organisation and United Nations Environment Programme. Blackwell, Oxford.

Signal Transduction Networks and the Integration of Responses to Environmental Stimuli

GARETH I. JENKINS

Plant Molecular Science Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, Bower Building, University of Glasgow, Glasgow G I 2 8QQ, UK

I. Introduction .......................................................................................................... A. Networks Versus Pathways .......................................................................... B. Achieving an 'Appropriate' Response .........................................................

54 55 56

11. Interactions Within Signalling Networks ............................................................. A. Evidence of Negative Regulation ..... B. Evidence for Synergism ...............................................................................

57 57 63

111. Approaches to Identify the Mechanisms Involved in Interactions Between Signalling Pathways .............................................................................................

67

Conclusions .......................................................................................................... Acknowledgements ............... ........................................................... References ............................. ......

69 70 70

IV.

This paper is dedicated to the late Professor Harold W. Woolhouse, who will be remembered as a great enthusiast for plant science. Professor Woolhouse was the author's PhD supervisor.

The survival of plants is dependent on their ahiliy to sense and respond appropriately to (I wide range of environmental stiniuli, some of which fire potentially harmful. Such responses often involve the expression of .spec@- sub-sets of genes whose products are concerned with rnininzi,-ing cell duniage. Each response must be uppropriute in the context of other respnnses rind the necessary coordination and integration of responses i s uchieved through interaction or 'cross-tulk' between the relevont signal transduction pathways. Exuinples ure discussed both of negative regulation between signalling pathways and of partirulur contbinations of priniar?,stimuli together eliciting a hyper-response. For instance, negative regulation is observed in the repression of photosynthetic genes b y sugars, in defence gene regulation and beMJeen phytochronie signal trunsduction piithwvays. In cvntrmt. srrong

54

G. 1. JENKINS

synergistic interactions are observed between, jor example, ethylene and methyl jasmonate in stimulating speciJc defence genes and between blue, UV-A and UV-B light in the regulation of chalcone synthase gene expression. The combined application of biochemical, molecular and genetic approaches offers the most powerful means of dissecting the cellular and molecular mechanisms involved in these interactions.

1. INTRODUCTION Being sedentary, plants are at the mercy of their environment. Throughout their growth and development they are exposed to a wide range of environmental variables. Although some of these, such as daylength, are benign and may be used as cues to initiate developmental transitions, others, such as extremes of temperature, drought, UV-B and pathogen attack are potentially very harmful. Plants have, therefore, evolved protective responses which minimize the adverse effects of these abiotic and biotic stresses. Many of the protective responses of plants involve differential gene expression. That is, particular subsets of genes are expressed in response to specific external stimuli. Some gene products are induced by a range of stimuli whereas others are much more specific. Over the last ten to fifteen years numerous genes have been identified that are responsive to particular stimuli. For example, low temperature stimulates the expression of over thirty different genes (Hughes and Dunn, 1996). Similarly, pathogen attack induces a range of genes, some of which encode proteins, such as glucanases and chitinases, that have antipathogenic effects, whereas others encode enzymes that synthesize compounds, ‘phytoalexins’, that limit pathogen invasion in the affected tissues (Cutt and Klessig, 1992; Dixon and Paiva, 1995). As a further illustration, exposure of plants to damaging UV-B radiation triggers the expression of genes encoding enzymes that synthesize UV-absorbing phenylpropanoid and flavonoid compounds in the epidermal tissues (Stapleton, 1992; Jenkins et al., 1997). It is evident that the survival of plants is dependent on their ability to sense and respond appropriately to a wide range of environmental stimuli. Hence, plant cells possess mechanisms to detect specific environmental signals, to transduce the information within the cells and to effect the appropriate responses. In the case of gene expression responses, the end point of signal transduction is likely to involve the activation of specific transcription factors, unless of course the gene in question is regulated entirely via post-transcriptional mechanisms. Transcriptional control itself is very complex. In addition to the reversible modification of transcription factors by, frequently, phosphorylation, there is evidence that transcription factors may be stimulated to move into or out of the nucleus as a result of modification (Harter et al., 1994; Terzaghi et al., 1997). Moreover, the initial effect of the external stimulus may be to elicit the synthesis of a transcription factor or other effector, which in turn stimulates the transcription of other genes involved in the response. Thus several gene expression responses are prevented by inhibitors of protein synthesis (Lam et al., 1989; Green and Fluhr, 1995; Christie and Jenkins, 1996).

SIGNAL TRANSDUCTION NETWORKS

55

Information on the signal transduction processes that mediate the effects of external stimuli on gene expression is gradually accumulating. Cell physiological techniques have identified events, such as transient increases in cytosolic calcium ion concentration, that are likely to be of key importance in mediating responses to, for example, mechanical stimulation (Knight et al., 1991), chilling (Knight et al., 1991, 1996) and light detected by phytochrome (Shacklock et al., 1992). Pharmacological approaches have further defined components of signalling pathways, for instance, initiated by UV-B and UV-Ahlue light (Chstie and Jenkins, 1996), phytochrome (Millar et al., 1994) and low temperature (Monroy and Dhindsa, 1995). In addition, the genetic approach has resulted in the identification and cloning of several novel signal transduction components, including those involved in light (Chory, 1993; Deng, 1994; Jenkins ef al., 1995), ethylene (Keiber, 1997) and abscisic acid (Merlot and Giraudat, 1997) signalling. These latter molecules, together with others such as jasmonic acid (JA) and salicylic acid (SA), are important in mediating responses to wounding and pathogen attack (ethylene, JA, SA) and low temperature and drought (abscisic acid). Research is now at the stage where the combined application of these complementary experimental approaches will result in a burgeoning of information on environmental signal transduction.

A.

NETWORKS VERSUS PATHWAYS

Signal transduction is often discussed in terms of ‘pathways’. Although this is a convenient and often appropriate way to refer to the processes through which a particular stimulus elicits a given response, it must be recognized that signal transduction may not proceed through a simple, linear sequence of events. Moreover, it is evident that multiple, potentially interacting, signal transduction pathways are present in cells. Clearly, the same stimulus may regulate several different processes and may do so via different or branching pathways. For example, the cold-induction of gene expression occurs through abscisic acid-dependent and -independent pathways (Nordin et al., 1991; Gilmour and Thomashow, 1991; Hughes and Dunn, 1996); also, phytochrome signal transduction involves distinct pathways (see section II.A.3). Furthermore, a particular process, such as the expression of a specific gene, may be regulated by a host of different stimuli. This is observed, for example, with the phenylalanine ammonialyase (PAL) and chalcone synthase (CHS) genes (van der Meer et al., 1993; Dixon and Paiva, 1995; Mol et al., 1996). It is therefore important to think in terms of ‘networks’ rather than isolated signalling pathways. There is increasing evidence that interactions occur within signal transduction networks and this will be discussed in more detail below. Thus signal transduction networks in cells contain overlapping, interconnected components. The perception of a given stimulus may therefore have an impact throughout the network, rather like the ripples formed when a stone hits a pond. Hence stimulus perception may

56

G. I. JENKINS

potentially affect the responses to other stimuli, whether these are perceived at the same time or subsequently. A further important point is that signal transduction networks are not identical in all cells and that the capacity for signalling may change during development. For instance, many cold-induced genes are expressed in shoot and not root tissues (Hughes and Dunn, 1996), indicating that signalling andor effector components of these responses are spatially expressed. An illustration of a temporal change in responsiveness is provided by the CHS genes in parsley and white mustard (Batschauer et al., 1991; Frohnmeyer et al., 1992). In these plants CHS expression is regulated by phytochrome early in leaf development but by UVhlue photoreceptors later in development. Similarly in Ambidopsis, phytochrome induction is lost in older leaves (Kaiser er al., 1995; Jackson et al., 1995). Again, changes in the capacity for signalling are likely to be involved. Hence, a challenge for future research is to understand the regulation of expression of signal transduction and effector components.

B. ACHIEVING AN ‘APPROPRIATE’ RESPONSE

Plants are continually bombarded with environmental information. It is therefore vital that each stimulus elicits an appropriate response. There will be a ‘cost’, in terms of resource expenditure in each response, so cumulative unnecessary responses would be ‘expensive’ and hence potentially damaging. Moreover, the inability to curtail a response when it is no longer needed would also consume finite resources. On the other hand, failure to respond urgently to a potentially damaging abiotic or biotic stress may prove lethal. It is therefore vital, in terms of competitiveness, genetic fitness and survival, that the responses to external stimuli are appropriate and measured. More precisely, each response should exhibit the required sensitivity to the stimulus (threshold of the dose-response relationship), rapidity, magnitude, duration and specificity (with regard to which genes are regulated). Furthermore, each response must be appropriate in the context of other responses. To achieve this, responses must be integrated. Thus, information from one stimulus may affect the response to another, perhaps to curtail the response or to amplify it. For instance, sudden exposure to a potentially lethal stimulus may require the diversion of resources into a protective response which takes priority over other responses. In this case, a stimulus may switch off some genes while switching on others. Alternatively, the presence of several stimuli together may reinforce the ‘message’ that the plant is being exposed to a particular environmental situation, more so than any of the stimuli alone would do. In this case, the combination of stimuli may elicit a synergistic response involving the hyperexpression of a battery of genes. Thus it is evident that mechanisms are required for information transfer, or cross-talk, between stimulus-response pathways. Such integration enables appropriate responses to be made.

SIGNAL TRANSDUCTION NETWORKS

57

This paper presents evidence for the existence of interactions between stimulusresponse pathways in plants. Examples are selected to illustrate particular points and it is not the intention to provide a comprehensive survey of the literature. The conclusion is that plant cells contain networks of potentially interacting signal transduction pathways and that these interactions enable the plant to integrate its responses to environmental stimuli to maximize its survival.

11. INTERACTIONS WITHIN SIGNALLING NETWORKS A.

EVIDENCE OF NEGATIVE REGULATION

In recent years evidence has been obtained that exposure to particular stimuli may result not only in the elicitation of a specific gene expression response, but the switching off of a separate response. Moreover, it has been shown that positive components of one signalling pathway may act as negative regulators of another. As discussed below, negative regulatory responses are of key importance both in the prioritization of responses to external stimuli and in achieving de-sensitization, the cessation of a response when it is no longer appropriate.

1. Negative regulation in the control of gene expression by metabolites It is vital for cells to regulate their metabolic activity in relation to both developmental and external factors. Particular organs and tissues constitute ‘sources’ or ‘sinks’ of metabolites and individual cells have a characteristic spectrum of metabolic activities commensurate with their roles in nutrient mobilization and biosynthesis. To illustrate, mature leaves function as net exporters of carbohydrates whereas developing seeds and meristems function as sinks. Metabolic activities within cells are regulated in the short term by the ‘fine control’ of enzyme activities, in particular through post-translational modification, and in the longer term by the ‘coarse control’ of gene expression. Environmental factors influence metabolism over both time scales through these processes. It is well established that the levels of particular metabolites, notably sugars, regulate the expression of various genes (Graham, 1996; Koch, 1996; Smeekens and Rook, 1997). Cells therefore possess mechanisms to sense the levels of specific metabolites and use this information to differentially regulate gene expression. However, many genes known to be regulated by metabolites are also regulated by environmental stimuli such as light or various stresses. Hence metabolic signalling processes must be integrated with environmental signal transduction pathways. A classic example of the metabolic regulation of gene expression is the repression of transcription of genes concerned with photosynthesis by soluble sugars such as glucose and sucrose. Sheen (1990) reported that the transcriptional activity of promoters of several genes encoding photosynthetic components was repressed by sugars in maize mesophyll protoplasts. Similarly, Krapp er al. (1993) found that the addition of glucose to a Chenopodium cell culture

58

G. 1. JENKINS

reduced expression of genes encoding the major chlorophyll-binding protein of thylakoids (CAB) and the small subunit (rbcS) of ribulose 1,5-bisphosphate carboxylase/oxygenase. Sugar repression of CAB transcripts was also observed in cultured Brassicu napus cells (Harter et al., 1993). Moreover, similar observations were reported when carbohydrate levels were artificially elevated in intact plants either by transgenic expression of apoplastic invertase (von Schaewen et al., 1990) or by cold-girdling to reduce source leaf export (Krapp and Stitt, 1995). The rbcS and CAB genes are induced by light in most higher plants and since, in the above examples, sugar repression was observed in the light, it can be concluded that the negative regulation by sugars overrides light signal transduction. Information is starting to accumulate regarding the signalling mechanisms involved in the sugar repression of gene expression. Experiments using glucose analogues and sugar metabolites led to the hypothesis that the phosphorylation of hexose sugars by hexokinase has a key role in sugar sensing (Graham el al., 1994; Jang and Sheen, 1994). Analogues of glucose which enter cells but are not metabolized by hexokinase, such as 3-O-methylglucose, did not repress rbcS expression in Chenopodium cells (Krapp et ul., 1993). Jang and Sheen (1994) reported similar effects on the repression of promoter-reporter fusions in maize protoplasts. The analogue 2-deoxyglucose, which can be phosphorylated by hexokinase but is not readily metabolized by glycolysis, was able to mediate repression. Furthermore, mannoheptulose, an inhibitor of hexokinase, blocked repression by 2-deoxyglucose. These findings point to hexokinase as the initiator of the signalling pathway resulting in sugar repression. Further support for this hypothesis was obtained in a recent study (Jang er al., 1997) with transgenic plants either under- or overexpressing hexokinase. Antisense reduction of hexokinase decreased glucose sensitivity of the plants in several responses whereas overexpression resulted in increased sensitivity. The hexokinase mechanism is not confined to photosynthetic genes, since sugar repression of genes encoding malate synthase and isocitrate lyase, which mobilize stored lipids following germination, also involves hexokinase (Graham et al., 1994). At present there is little detailed information on the sugar signalling pathway mediated by hexokinase and it is not known how this signalling pathway negatively regulates light signal transduction. Further information on sugar sensing is likely to be obtained from comparative studies with yeast, which also has a hexokinasemediated sugar sensing system. For example, a kinase termed SNFl is important in the yeast system and plant homologues of SNFl have been identified that can complement snfl mutants (Muranaka et al., 1994). There may also be parallels with sugar sensing in mammalian cells, which involves a glucokinase activity. Urwin and Jenkins (1997) reported that a promoter element involved in sugar repression of a Phaseolus vulgaris rbcS gene resembles elements concerned with the induction of mammalian genes by sugars. Although the above discussion has focused on the repression of genes by sugars, it should be mentioned that there are several examples of plant genes which are stimulated by sugars. These include CHS, nitrate reductase, patatin, sporamin and

SIGNAL TRANSDUCTION NETWORKS

59

Phenylalanine PAL Cinnarnate

/

4-Cournaroyl-CoA

CHS

Naringenin chalcone

FLAVONOIDS ANTHOCYANINS

Fig. 1. Phenylpropanoid and flavonoid biosynthesis. The general phenylpropanoid pathway includes the steps from phenylalanine to 4-coumaroyl-CoA and is initiated by PAL (phenylalanine ammonia-lyase). CHS (chalcone synthase) catalyses the first step in flavonoid biosynthesis, the formation of naringenin chalcone. Further enzymatic steps (not shown) lead to the formation of specific flavonoids, anthocyanins, sinapic acid esters and furanocoumarins. For information on other branches of the pathways see Dixon and Paiva (1995).

P-amylase (Graham, 1996; Koch, 1996; Smeekens and Rook, 1997). In some cases (nitrate reductase: Jang et al., 1997; CHS: Urwin and Jenkins, 1997) there is evidence consistent with a hexokinase signalling mechanism. Therefore this sugar sensing system appears to be able to mediate both the repression and induction of gene expression.

2. Negative regulation in plant defence responses It is well established that genes encoding enzymes of the phenylpropanoid and flavonoid biosynthetic pathways are responsive to a range of environmental stimuli (van der Meer et al., 1993; Dixon and Paiva, 1995; Mol et al., 1996). PAL is the first enzyme in the general phenylpropanoid pathway and CHS is the first committed step in the branch from the pathway that leads to flavonoid biosynthesis (Fig. 1). Various products of these pathways have a key role in limiting the damaging effects of abiotic and biotic stresses. Both sinapic acid esters, derived from the phenylpropanoid pathway, and particular flavonoids function as UV-protectants in the epidermal layers (Stapleton, 1992; Jenkins er al., 1997). Compounds such as furanocoumarins, again derived from the phenylpropanoid pathway, have antimicrobial activity and are therefore important in defence against pathogens (Hahlbrock and Scheel, 1989; Hahlbrock et al., 1995; Dixon and Paiva, 1995).

60

G . 1. JENKINS

The regulation of transcription of PAL and CHS genes in response to various stimuli has been studied extensively in several species (van der Meer et al., 1993; Dixon and Paiva, 1995; Mol et al., 1996). In parsley, as in other species, both PAL and CHS genes are induced by UVhlue light. However, PAL expression is strongly induced by an elicitor derived from the fungus Phytophthora megasperma, whereas CHS expression is not (Lozoya et al., 1991). Furthermore, enzymes of the branch from the phenylpropanoid pathway that leads to furanocoumarin biosynthesis are stimulated by elicitor and not by light. Thus, it can be demonstrated that elicitor treatment of cultured parsley cells leads only to furanocoumarin, and not flavonoid, accumulation and that UV light has the opposite effect. Lozoya et al. (1991) demonstrated that flavonoid accumulation and the induction of CHS expression in response to UV light is in fact prevented by the fungal elicitor. In contrast, furanocoumarin accumulation and the induction of PAL expression by the elicitor was not switched off by UV light, although there was a significant reduction. The negative regulation of CHS expression by elicitor treatment was at the level of transcription. Although the mechanism of repression is not known, it most likely involves signalling events which affect the biogenesis and/or activation of transcription factors that associate with cis-elements in the CHS promoter, There is evidence that the UV light stimulation of CHS transcription requires both protein synthesis (Christie and Jenkins, 1996) and the posttranslational activation of relevant transcription factors (Harter et al., 1994). It is likely that the repression of CHS by the fungal elicitor serves to divert metabolites into the biosynthesis of compounds which are important in the defence response. Hence, negative regulation enables the plant to prioritize its protective responses to potentially damaging stimuli. It appears that pathogen attack requires a more urgent response than protection against UV radiation, although whether a different response would be observed to more severe UV-B exposure or to different pathogen signals is not known. Lozoya et al. (1991) suggested that negative regulation may be a common phenomenon in plant defence responses and cited several examples where elicitors differentially regulate biosynthetic processes. In fact recent work by Vidal et al. (1 997) illustrates that factors regulating defence gene expression may have mutually antagonistic effects. These authors studied the induction of several genes encoding pathogenesis-related (PR) proteins in tobacco in response to the soft-rot pathogen Erwinia carotovora. PR proteins are induced by a range of pathogens. They have been classified into various groups and several have been shown to have antipathogenic properties (Cutt and Klessig, 1992). Erwinia produces extracellular enzymes that generate elicitors by digestion of the host cell wall and the elicitors induce gene expression both at the local site of infection and systemically in other leaves of the plant. Hence a culture filtrate of Erwinia containing the enzymes is effective in inducing the defence response. In this case transcripts of a basic glucanase and of acidic and basic chitinase genes were induced rapidly in the plants in both treated and non-treated leaves. SA, which is known to be an important signalling molecule in the production of systemic resistance, induced the same

SIGNAL TRANSDUCTlON NETWORKS

61

genes more slowly and weakly, indicating that another signal was involved in their rapid induction in non-inoculated leaves. In contrast SA, but not the culture filtrate, induced the P R - l a gene. Thus, in common with other systems, there appear to be distinct pathways for the induction of defence genes in tobacco in response to infection by a particular pathogen. Vidal rt al. (1997) further showed that treatment with increasing amounts of the Erwinia culture filtrate negatively regulated the induction of the PR-la gene in response to SA. Similarly, SA inhibited the induction of the basic glucanase gene by the culture filtrate. The authors suggested that such reciprocal antagonistic effects may be mediated by signalling components common to different defence pathways which could function as either positive or negative regulators of gene expression. Such a mechanism would provide an efficient way for the plant to coordinate its local and systemic defences in response to attack by different pathogens. Identification of components of the pathways will enable this hypothesis to be tested. Negative regulation between phytochrome signal transduction pathways A further illustration of negative regulation is provided by the phytochrome signal transduction pathways controlling gene expression (Bowler and Chua, 1994; Millar et a1 1994). Phytochrome stimulates the expression of a range of plant genes, including the CHS genes and those encoding various chloroplast proteins, such as CAB. In recent years, information on the components of phytochrome signal transduction pathways has been obtained using direct microinjection of molecules into plant tissue and from pharmacological studies with cultured cells. Neuhaus et al. (1993) showed that microinjection of phytochrome (as phytochrome A) into hypocotyl subepidermal cells of the phytochrome deficient tomato nurea mutant restored both the phytochrome-mediated induction of chloroplast development and anthocyanin accumulation. Anthocyanin is a product of the flavonoid biosynthesis pathway (Fig. 1) and it was demonstrated that phytochrome microinjection stimulated expression of a gene fusion consisting of a CHS promoter ligated to the P-glucuronidase (GUS) reporter gene. Similarly, phytochrome injection stimulated the promoter of the CAB gene. By co-injecting various putative signal transduction components, Neuhaus et nl. ( 1993) and Bowler et al. (1994a) provided evidence that the phytochrome stimulation of both promoters required one or more heterotrimeric G-proteins. Furthermore, CHS promoter activity and anthocyanin accumulation were stimulated by cyclic GMP (cGMP). In contrast cGMP did not initiate chloroplast development. However, CAB promoter activity and the production of chlorophyll-containing plastids was stimulated by the injection of Ca'+ ions and the regulatory calcium-binding protein calmodulin. Neither Ca'+ nor calmodulin activated the CHS promoter or anthocyanin accumulation. These data therefore provide evidence for separate phytochrome signal transduction pathways with distinct target genes (Fig. 2). The pathways involve a G-protein as an early step and then bifurcate, one branch involving cGMP and stimulating

62

G . 1. JENKINS

7 GEN

Pfr

G Protein

f \

cGMP

CHS

FNR

CAB

Fig. 2. Interactions between phytochrome signal transduction pathways. Phytochrome Pfr activates a G protein and the pathways then bifurcate, one branch involving cGMP and stimulating CHS expression and the other involving cytosolic Ca2+and calmodulin (Cah4) and stimulating CAB expression. Both pathways are required to stimulate FNR expression. Inhibition of the cGMP pathway by genistein (GEN) is shown. The dashed lines indicate reciprocal negative regulation of the pathways. Modified from Bowler and Chua (1994).

CHS and one involving Ca2+/calmodulin and stimulating CAB. The authors further demonstrated that the production of chloroplasts with photosystem I and cytochrome b6f components, and also the expression of genes encoding particular PSI proteins (e.g. the ferredoxin NADP' oxidoreductase, FNR), required both signalling pathways (Fig. 2). That is, the production of chloroplasts with a complete set of complexes was stimulated by injection of cGMP and Ca2'/calmodulin together, but by neither compound alone. To complement the microinjection experiments, Bowler et al. (1994a) showed that cell-permeable cGMP analogues stimulated CHS transcript accumulation, but not CAB or FNR gene expression, in darkness in a soybean cell culture, Bowler et al. (1994b) provided evidence that the two distinct phytochrome signal transduction pathways were subject to reciprocal negative regulation. That is, components that had a positive function in one signalling pathway mediated a repressive effect on the other. Microinjection of high cGMP concentrations caused repression of CAB-GUS gene expression induced by Ca*+/calmodulin. Similarly, introduction of cGMP into soybean cells attenuated the level of CAB transcripts. The induction of CHS in soybean cells, and of the CHS-GUS fusion in injected tomato hypocotyl cells, was inhibited by the histidine/tyrosine kinase inhibitor genistein. The target of this compound was found to be downstream of cGMP in the signalling pathway. Genistein prevented the repression of CAB expression by cGMP, indicating that the component that interacts with the Ca2'/calmodulin pathway is likely to be downstream of cGMP and is either sensitive to genistein or downstream of the genistein-sensitive component. Interestingly, the light-induction of CHS expression in the soybean cells was transient, peaking after about 3 h. Thus there appeared to be desensitization to the stimulus during prolonged exposure. This phenomenon was not observed when cGMP was applied to dark-treated cells, indicating that transient levels of cGMP may be the cellular basis of stimulation followed by desensitization during normal illumination. cGMP therefore appears to regulate both phytochrome signalling pathways.

SIGNAL TRANSDUCTION NETWORKS

63

Evidence that the Ca’+/calmodulin-dependent pathway could negatively regulate the cGMP pathway was obtained from experiments with inhibitors of CAB expression. Several compounds, including calcium channel and calmodulin inhibitors, prevented the light induction of CAB in soybean cells and the phytochrome A induction of CAB-GUS expression in microinjected tomato cells. However, these inhibitors additionally caused a hyperinduction of CHS transcript levels in the cell culture. In addition, Bowler et al. (1994b) found that injection of high concentrations of Ca”/calmodulin into tomato cells inhibited CHS-GUS expression stimulated by a constant level of co-injected cGMP. Thus Ca’‘ and calmodulin directly, or via a downstream component, function to negatively regulate the cGMP dependent phytochrome signalling pathway. The effect appears to be specifically on the extent of induction of CHS, because desensitization remains in the presence of the inhibitors of the Ca’+/calmodulin pathway. Direct measurements of the in vivo levels of cGMP and Ca2’ in the tomato and soybean systems are now required to complement the above findings. These will give more detailed information on the cellular mechanisms involved in the transduction of phytochrome signals. The above studies provide valuable insights into the cellular mechanisms of negative regulation of specific signalling pathways in plant cells. Bowler et a / . (1994b) speculated on the significance of these intriguing control mechanisms. They suggested that negative regulation could function to promote the synthesis of photoprotective compounds by the flavonoid biosynthesis pathway when plants are first exposed to light and to prevent the development of functional complexes in chloroplasts until the photoprotective mechanisms were in place.

B. EVIDENCE FOR SYNERGISM

In contrast to the above examples of negative regulation, there are several instances in the literature of synergistic interactions between signalling pathways resulting in hyper-responses to particular combinations of stimuli. In some cases, such as the interaction between NaCl and abscisic acid (ABA) in the regulation of Em gene expression in rice (Bostock and Quatrano, 1992). each stimulus elicits a significant response, although their combined effect is larger than the addition of their separate effects. In other cases, such as the interaction between methyl jasmonate (MeJA) and sugars in the expression of vegetative storage protein genes in soybean (Mason et al., 1992), the separate stimuli give negligible responses but hyper-responses when together. In some cases a stimulus which is not effective on its own may permit or enhance a response to another stimulus. For instance, blue light induces various responses in higher plants which are not elicited by red light, detected by phytochrome; however, in several cases illumination with far-red light prevents the blue light response, indicating that phytochrome in the Pfr form is required for blue light to be effective (Mohr, 1994). Synergistic responses reveal the existence of important mechanisms which

64

G . 1. JENKINS

ensure that plants respond appropriately to particular environmental conditions. Considering all the environmental information that impinges on a plant, it is important to have a means of discriminating, of being able to recognize when environmental conditions necessitate a particularly urgent, or especially large response. This is achieved by particular combinations of stimuli together eliciting much greater responses than those induced by individual stimuli. The examples discussed below illustrate the importance of synergism in the protection of plants against biotic and abiotic stresses.

I . Synergistic interactions regulate plant defence genes It is well known that exposure of plants to pathogens elicits the expression of a battery of genes. Prominent among the proteins synthesized are the PR proteins (see section II.A.2). Several of the signalling molecules involved in the regulation of particular PR genes have been identified and among these are SA (see section ILA.2), ethylene and JA. Xu et al. (1994) studied the regulation of two PR protein genes in tobacco, encoding PR- 1 and osmotin, a PR-5 protein. The latter protein owes its name to the fact that it accumulates in response to salt (NaCI) stress (Singh et al., 1987).In fact Osmotin gene expression is induced by a range of external and endogenous signals (Cutt and Klessig, 1992). Xu et al. (1994) reported that ethylene stimulated the Osmotin promoter, fused to GUS, in transgenic tobacco, whereas treatment with MeJA did not. However, a combination of ethylene and MeJA induced a very much larger increase than that seen with either stimulus. The increase was more than additive and therefore indicated a synergistic interaction between the ethylene and MeJA signalling pathways. Similar results were observed at the levels of mRNA and protein accumulation. The P R - l b gene showed minimal expression in response to either ethylene or MeJA, but again a large synergistic increase was observed in the presence of both compounds. Synergism in P R - l b expression was also observed between SA and MeJA. Interestingly, the extent of interaction between ethylene and MeJA in stimulating Osmotin gene expression differed between organs of the plant. In this case, a difference was observed between roots and cotyledons. Roots showed a much higher level of Osmotin promoter activity than cotyledons in control, non-treated seedlings. Both organs showed minimal stimulation of the Osmotin promoter by MeJA above the control level and a substantial ethylene stimulation. Evidence of synergism was observed between MeJA and ethylene in roots, but in cotyledons the hyperstimulation of the promoter was much greater than in roots, with approximately a 200-fold increase in GUS activity. Although differences in the endogenous levels of ethylene and MeJA in non-stimulated tissue may in part explain these findings, it is likely that there are genuine organ-specific differences in responsiveness. This emphasizes a point made earlier, that components of signalling networks may themselves be subject to spatial, temporal or environmental regulation. Xu et al. (1994) pointed out the significance of the hyperinduction of defence gene expression by combinations of signals. In the case of a gene (such as encoding

SIGNAL TRANSDUCTION NETWORKS

65

osmotin) regulated by several, apparently unrelated signals, the particular combination of signals, rather than any one signal alone, may be the key inductive stimulus. The synergistic hyperinduction of various defence genes resulting from the combined presence of several defence-associated signals may therefore be of key significance in vivo. An illustration of the significance of such synergistic interactions in plant defence is provided by the recent work of Shirasu et al. (1997). These authors examined the role of SA in inducing local, as opposed to systemic, defence responses. Pathogen attack often elicits a localized ‘hypersensitive response’ (HR) which involves the production of superoxide radicals and hydrogen peroxide in the ‘oxidative burst’ and the activation of defence genes. Superoxide is a component of the signalling pathway that promotes defence gene activation (Jabs et al., 1997). Hydrogen peroxide has antimicrobial effects and, in addition, promotes oxidative cross-linking which strengthens the cell wall, causes localized cell death and stimulates the expression of several protective genes (Bradley el al., 1992; Levine et al., 1994; Jabs et al., 1997; Lamb and Dixon, 1997). The HR is a key element in plant defences against pathogen attack because it helps to prevent the spread of the pathogen. Exogenous SA, when applied alone, is often required at relatively high concentrations or for extended periods to induce defence responses. However, Shirasu et al. (1997) found that SA at low, physiological concentrations acted synergistically with a pathogen to induce a rapid localized defence response in soybean cells. In these experiments a strain of Pseudonionas syringae pv. glycinea was used that elicited a HR in the chosen soybean genotype. SA, in the presence of the pathogen, caused the production of high levels of hydrogen peroxide within two hours; levels much greater than those seen with either SA or the pathogen alone over the equivalent period. Moreover, the combination of SA and the pathogen accelerated hypersensitive cell death and the induction of genes encoding PAL and glutathione S-transferase. Thus SA, through its synergistic interaction with the pathogen, has an important role in potentiating the rapid localized response to pathogen attack.

2. Synergistic interaction between UV and blue light stimuli in the regulation of CHS expression Returning to the regulation of flavonoid biosynthesis genes, Fuglevand et al. (1996) have demonstrated that complex interactions between UV and blue light signal transduction pathways result in hyperstimulation of CHS expression in Arabidopsis. In Arabidopsis the phytochrome regulation of CHS expression is restricted to very young seedlings. In light-grown leaf tissue, induction is predominantly by separate UV-B and UV-A/blue light signal transduction pathways, the latter coupled to the CRY 1 photoreceptor (Fuglevand et al., 1996). The UV-B and UV-A/blue light signal transduction pathways are distinct from those involved in phytochrome regulation (Christie and Jenkins, 1996). Fuglevand et a/. ( I 996) reported experiments with transgenic Arabidopsis plants

66

G . I. JENKINS

containing a CHS promoter-GUS fusion. Plants were grown for several weeks in a low fluence rate of white light that did not stimulate significant CHS-GUS expression. Subsequent exposure of mature leaves to either UV-B, UV-A or blue light resulted in approximately a 10-fold stimulation of CHS-GUS expression and similar increases were observed in the level of CHS transcripts. The authors categorized these responses to individual light qualities as ‘inductive’ responses. Synergistic responses were observed in specific combinations of light qualities. Exposure to either UV-B and blue light, or UV-B and W - A light, given simultaneously, resulted in approximately 4- to 8-fold greater stimulation of CHS-GUS expression than in the inductive treatments. UV-A and blue light gave an additive rather than a synergistic effect. Fuglevand et al. (1996) further demonstrated that blue light given before UV-B resulted in a synergistic response whereas UV-B before blue light did not. This indicated that blue light generates a signal or activates a component that interacts productively with components of the UV-B pathway to elevate the response. UV-B itself was unable to produce such an effect. When a dark period intervened between the blue and subsequent UV-B exposure, the ‘synergism signal’ was gradually lost. Nevertheless, the signal was sufficiently stable in darkness even after 15 h to give an enhanced response in subsequent UV-B compared to a control that had received no blue preillumination. In contrast, UV-A treatment prior to UV-B did not result in a synergistic enhancement of CHS expression and neither did UV-B exposure before UV-A. The fact that marked synergism was nevertheless observed with both treatments given simultaneously indicates that the signal generated by UV-A is not stable, but transient. Fuglevand et al. (1996) therefore concluded that the pathways producing the signals in blue and UV-A light were distinct. Consistent with this finding, they further showed that the CRY1 photoreceptor was not involved in the synergistic responses, because both synergisms were retained in the hy4-2.23N mutant, which lacks the CRY 1 photoreceptor. The different pathways involved are shown in Fig. 3. Further experiments showed that the distinct ‘synergism signals’ generated by blue and UV-A light could interact together with the UV-B signal transduction pathway to maximize the level of CHS expression. Plants were exposed to blue light and then to UV-A plus UV-B. In this case, the level of CHS-GUS expression observed was approximately double that produced with either synergistic combination alone. The results therefore indicated that the two synergistic interactions could function in an additive manner to maximize CHS expression. In fact, expression was 150-fold compared to the 10-fold increase observed with the single light qualities. This is the first report of two synergistic interactions together stimulating expression of the same gene. So what is the significance of the above synergistic interactions to the plant? Of course, plants are not exposed to separate UV and blue light qualities but to a complex spectrum. It is possible that seedlings growing under a leaf canopy may not experience UV-B radiation until they have been exposed to longer wavelengths, and some degree of potentiation of the response by blue light could therefore occur.

SIGNAL TRANSDUCTION NETWORKS

67

UV-Nblue blue

blue

I

I b

\stable \

UV-B

’.-.--’CHS /*

:tansient

I

I

UV-A

Fig. 3. Signal transduction pathways involved in the regulation of CHS gene expression by UV and blue light in Arubidopsis. Inductive pathways, involving either CRY I or the UV-B light detection system, are shown by solid lines. An additional, hypothesized inductive blue light signalling pathway is indicated by a dot-dash line. The distinct UV-A and blue light pathways that interact synergistically with the UV-B pathway and produce transient and relatively stable signals respectively, are represented by dashed lines. No information is available on the specific sites of interaction of the synergism pathways with the UV-B pathway. Reproduced from Fuglevand et al. (1996) with permission.

However, UV-B exposure should always be accompanied by UV-A and blue wavelengths. Hence the synergistic interactions will be the norm. The maximal stimulation of CHS expression will provide a basis for the synthesis of high levels of protective flavonoid pigments. However, it is not yet known whether other genes encoding flavonoid biosynthesis enzymes are subject to the synergistic regulation. Fuglevand el al. ( 1996) reported that the synergistic interactions do appear to have selective value. Aruhidopsis plants exposed to 24 h of UV-B radiation alone normally died whereas those exposed to combinations of UV-B and UV-A and/or blue light did not.

111. APPROACHES TO IDENTIFY THE MECHANISMS INVOLVED IN INTERACTIONS BETWEEN SIGNALLNG PATHWAYS To understand how plant cells regulate key genes in response to environmental stimuli, and how these responses are integrated, it is essential to identify components of the relevant signalling pathways and to define their specific functions. Furthermore, it is essential to identify the transcription factors that effect particular responses and to understand how terminal components of the signalling pathways, most likely protein kinases and phosphatases, regulate these effectors. Different signalling pathways may converge on the same transcription factor targets or, alternatively, different responses may be achieved through response-specific effectors. Establishing the specificity of function of particular components, such as transcription factors and kinases, will help us to understand how cells selectively stimulate transcription of specific genes in response to particular stimuli.

68

G.I. JENKINS

The most powerful way to identify signal transduction and effector components is to use a combination of cell physiological, biochemical, molecular and genetic approaches. The cell physiological and biochemical approaches being used with cell cultures, microinjection systems and transgenic plants will be important in identifying components involved in cross-talk between signalling pathways. For example, pharmacological approaches combined with microinjection have already provided valuable insights into the mechanisms of negative control operating between phytochrome signalling pathways (Bowler el a/., I994b). Transgenic plants used in conjunction with the hy4 mutant have facilitated the analysis of synergism in the UV and blue light regulation of CHS expression (Fuglevand et al., 1996). Since calcium is a key second messenger in several of these responses, the use of transgenic plants expressing calcium-sensitive aequorin fusions targeted to particular cellular compartments (Knight et ul., 1996) will enable investigation of the roles of different calcium pools in the interactions between signalling pathways. In most cases, the functions of individual transcription factors in responses to external stimuli have not been defined. Although nuclear protein-promoter DNA binding activities have been described for various genes, it is usually unknown which member of a family of transcription factors interacts with a specific promoter element in vivo to effect a particular response. Moreover, relatively little progress has been made in identifying specific kinases, phosphatases and other components which couple second messengers to transcription factors. The genetic approach will be particularly important in this respect. The isolation and characterization of mutants altered in the environmental regulation of particular genes will enable the functions of specific transcription factors, protein kinases and phosphatases in these responses to be defined. This is proving to be the case in studies, for example, of ethylene and abscisic acid signal transduction, in which mutants have facilitated the isolation of novel signalling components (Bowler and Chua, 1994; Keiber, 1997; Merlot and Giraudat, 1997). Thus CTRl encodes a raf-like kinase (Keiber ef al., 1993), ABIl and AB12 encode protein phosphatases (Leung et al., 1997) and AB13 encodes a putative transcription factor (Giraudat et al., 1992). Furthermore, by the use of appropriate screens, it should be possible to identify components which mediate interactions between signalling pathways. To date, no plant mutants have been found that are altered in components which function specifically in cross-talk between signalling pathways. Transgene expression screens provide the best means of identifying components which couple specific signals to defined promoter targets. Several mutants altered in the light-regulation of CAB (Li et al., 1994, 1995; Millar et al., 1995) and CHS (Jackson el al., 1995) genes have now been isolated using this approach. The availability of these mutants will enable the corresponding wild-type genes to be cloned using the powerful techniques developed for this purpose in Ambidopsis. In addition, the construction of double mutants will enable the functional interactions between the corresponding genes to be investigated. This type of analysis has proved very effective, for example, in studies of seedling responses to light; good testable models have been

SIGNAL TRANSDUCTION NE'IWORKS

69

developed hypothesizing the order of gene function and the relationships between pathways coupled to different photoreceptors (Chory, 1993). Interestingly, several of the putative signal transduction mutants isolated to date show essentially constitutive responses in the absence of a stimulus, for example, the cop/det@us mutants which are de-etiolated in darkness (Chory, 1993; Deng, 1994; MisCra et al., 1994) and c f r l , which has a constitutive response to ethylene (Keiber er al., 1993; Keiber, 1997). These mutants therefore identify negative regulators which constrain the response of the wild-type. The Ambidopsis icxl mutant also identifies a negative regulator, but is a different class of mutant in that it shows an enhanced response to the stimulus, in this case elevated expression of flavonoid biosynthesis genes in response to light (Jackson et al., 1995). The frequency of negative regulator mutants may reflect the nature of the screens used to isolate them or may indicate the widespread significance of such regulators in plant signal transduction. The latter point is emphasized by Bowler and Chua (1994). Negative regulators are certainly likely to be important in the interactions between signalling pathways, both in effecting repression and in synergistic responses. Although signal transduction pathways evidently generate positive signals which promote transcription, it is reasonable to hypothesize that the extent and speed of the response is often constrained by negative regulators associated with specific signalling pathways. Particular stimuli, or combinations of stimuli, may remove these constraints by inactivating specific negative regulators. Thus, synergistic interactions may represent the removal of negative regulation in parallel with the production of a positive signal. In other instances, for example in the interaction between the phytochrome signalling pathways or the effect of fungal elicitor on CHS expression, a signalling component that acts as a positive element in one pathway may function as a negative regulator of another.

IV. CONCLUSIONS Research is starting to elucidate the signal transduction processes in plants that couple external stimuli to transcription. It is evident that signals are not perceived and transduced in isolation. There are mechanisms for interaction or cross-talk within signal transduction networks which achieve the necessary coordination and integration of responses. There is evidence both of negative regulation between pathways and of particular combinations of primary stimuli together eliciting a hyper-response. In addition, responses to environmental stimuli must be integrated with other cellular activities; thus particular genes may be regulated by endogenous metabolic and developmental signals as well as by external signals. The cellular and molecular mechanisms which effect the different transcriptional responses to diverse stimuli are therefore likely to be extremely complex. In recent years the application of complementary experimental approaches has started to generate important information on the mechanisms of primary signalling processes as well as on the interactions between signalling pathways. In particular,

70

G.I. JENKINS

the ability to make direct measurements of signalling processes, such as the activities of ion channels and transient increases in cellular calcium concentrations has resulted in major advances. So too has the development of systems enabling the direct introduction of signalling components by microinjection and the pharmacological analysis of signal transduction in cell cultures. In parallel, the genetic approach has identified novel components of signalling pathways. These approaches need increasingly to be integrated. Furthermore, the application of these approaches to the components that mediate specifically the interactions between signalling pathways will be instrumental in elucidating the cellular and molecular mechanisms involved.

ACKNOWLEDGEMENTS The author is indebted to the UK Biotechnology and Biological Sciences Research Council and the Gatsby Charitable Foundation for the support of his research on signal transduction.

REFERENCES Batschauer, A., Ehmann, B. and Schiifer, E. (1991). Cloning and characterisation of a chalcone synthase gene from mustard and its light-dependent expression. Plant Molecular Biology 16, 175-1 85. Bostock, R. M. and Quatrano, R. S . (1992). Regulation of Em gene expression in rice: interaction between osmotic stress and abscisic acid. Plant Physiology 98, 1 3 5 6 1363. Bowler, C. and Chua, N-H. (1994). Emerging themes of plant signal transduction. Plant Cell 6, 1529-1541. Bowler, C., Neuhaus, G., Yamagata, H. and Chua, N-H. (1994a). Cyclic GMP and calcium mediate phytochrome phototransduction. Cell 77, 73-8 1. Bowler, C., Yarnagata, H., Neuhaus, G. and Chua, N-H. (1994b). Phytochrome signal transduction pathways are regulated by reciprocal control mechanisms. Genes and Development 8, 2 188-2202. Bradley, D. J., Kjellbom, P. and Lamb, C. J. (1992). Elicitor- and wound-induced oxidative cross-linking of a proline-rich plant cell wall protein: a novel, rapid defense response. Cell 70, 21-30. Chory, J. (1993). Out of darkness: mutants reveal pathways controlling light-regulated development in plants. Trends in Genetics 9, 167-172. Christie, J. M. and Jenkins, G. I. (1996). Distinct W - B and UV-A/blue light signal transduction pathways induce chalcone synthase gene expression in Arabidopsis cells. Plant Cell 8, 1555-1567. Cutt, J. R. and Klessig, D. F. (1992). Pathogenesis-related proteins. In “Genes Involved in Plant Defense” (T. Boller and F, Meins, eds) pp. 209-243. Springer-Verlag, New York. Deng, X-W. (1994). Fresh view of light signal transduction in plants. Cell 76, 423426. Dixon, R. A. and Paiva, L. A. (1995). Stress-induced phenylpropanoid metabolism. Plant Cell 7 . 1085-1097.

SIGNAL TRANSDUCTION NETWORKS

71

Frohnmeyer, H., Ehmann, B., Kretsch, T., Rocholl, M., Harter., K., Nagatani, A., Furuya, M., Batschauer, A., Hahlbrock, K. and Schafer, E. (1992). Differential usage of photoreceptors for chalcone synthase gene expression during plant development. Plant Journal 2, 899-906. Fuglevand. G., Jackson, J. A. and Jenkins, G. I. (1996). UV-B, UV-A and blue light signal transduction pathways interact synergistically to regulate chalcone synthase gene expression in Arabidopsis. Plant Cell 8, 2347-2357. Gilmour, S. J. and Thomashow, M. E (1991). Cold acclimation and cold-regulated gene expression in ABA mutants of Arabidopsis thalinna. Plant Molecular Biology 17, 1233-1 240. Giraudat, J., Hauge, B. M., Valon, C., Smalle, J., Parcy, F. and Goodman, H. M. (1992). Isolation of the Arabidopsis ABI3 gene by positional cloning. Plant Cell 4, I25 1-1261. Graham, I. A. (1996). Carbohydrate control of gene expression in higher plants. Research in Microbiology 147. 572-580. Graham, I. A., Denby, K. J. and Leaver, C. J. (1994). Carbon catabolite repression regulates glyoxylate cycle gene expression in cucumber. Plant Cell 16, 761-772. Green, R. and Huh,R. (1995). UV-B-induced PR-I accumulation is mediated by active oxygen species. Plant Cell 17, 203-212. Hahlbrock, K. and Scheel, D. ( 1989). Physiology and molecular biology of phenylpropanoid metabolism. Annual Review of Plant Ph-wiology and Plant Molecular Biology 40, 347-369. Hahlbrock, K., Scheel, D., Logemann, E., Numburger, T., Pdrniske, M.. Reinold, S., Sacks, W. R. and Schmelzer, E. ( 1995). Oligopeptide elicitor-mediated defense gene activation in cultured parsley cells. Proceedings of the National Academy of Science (USA) 92, 41504157. Harter, K., Talke-Messerer, C., Barz, W. and Schafer, E. (1993). Light- and sucrose-dependent gene expression in photomixotrophic cell suspension cultures and protoplasts of rape (Brassica napus L.). Plant Journal 4, 507-516. Harter, K., Kircher, S., Frohnmeyer, H., Krenz, M., Nagy, F. and Schlfer, E. (1994). Light-regulated modification and nuclear translocation of cytosolic G-box binding factors in parsley. Plant Cell 6, 545-559. Hughes, M. A. and Dunn, M. A. (1996). The molecular biology of plant acclimation to low temperature. Journal of Experimental Botany 47, 29 1-305. Jabs, T., Tschope, M., Colling, C., Hahlbrock, K. and Scheel, D. (1997) Elicitor-stimulated ion fluxes and 0; from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley. Proceedings of rhe National Academy of Science (USA) 94, 48004805. Jackson, J. A., Fuglevand, G., Brown, B. A., Shaw, M. J. and Jenkins, G. I. (1995). Isolation of Arabidopsis mutants altered in the light-regulation of chalcone synthase gene expression using a transgenic screening approach. Plant Journal 8, 369-380. Jang, J-C. and Sheen, J. (1994). Sugar sensing in higher plants. Plant Cell 6, 1665-1679. Jang, J-C., Leon, P., Zhou, L. and Sheen, J. (1997). Hexokinase as a sugar sensor in higher plants. Plant Ce6l 9, 5-19. Jenkins, G. I., Christie, J. M., Fuglevand, G., Long, J. C. and Jackson, J. A. (1995). Plant responses to UV and blue light: biochemical and genetic approaches. Plunt Science 112, 117-1 38. Jenkins, G. I., Christie, J. M., Fuglevand, G. (1997). UV-B perception and signal transduction. In “Plants and UV-B, Society for Experimental Biology Seminar Series 64” (P. J. Lumsden, ed.) pp. 135-156. Cambridge University Press, Cambridge, UK. Kaiser, T., Emmler, K., Kretsch, T., Weisshaar, B., Schafer, E. and Batschauer, A. (1995).

72

G.1. JENKINS

Promoter elements of the mustard CHSl gene are sufficient for light-regulation in transgenic plants. Plant Molecular Biology 28, 2 19-229. Keiber, J. J. (1997). The ethylene response pathway in Arabidopsis. Annual Review ofPlunt Physiology and Plant Molecular Biology 48, 277-296. Keiber, J. J., Rothenberg, M., Roman, G., Feldmann, K. A. and Ecker, J. R. (1993). CTRI, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases. Cell 72, 4 2 7 4 1 . Knight, H., Trewavas, A. J. and Knight, M. R. (1996). Cold calcium signalling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. Plan? Cell 18, 489-503. Knight, M. R., Campbell, A. K., Smith, S . M. and Trewavas, A. J. (1991). Transgenic plant aequorin reports the effect of touch and cold shock and elicitors on cytoplasmic calcium. Nature 352, 524-526. Koch, K. E. (1996). Carbohydrate-modulated gene expression in plants. Annual Review of Plant Physiology and Plant Molecular Biology 47, 509-540. Krapp, A. and Stitt, M. (1995). An evaluation of direct and indirect mechanisms for the “sink-regulation” of photosynthesis in spinach: Changes in gas exchange, carbohydrates, metabolites, enzyme activities and steady-state transcript levels after cold-girdling source leaves. Plunra 195, 3 13-323. Krapp, A., Hofmann, B., Schafer, C. and Stitt, M. (1993). Regulation of the expression of rbcs and other photosynthetic genes by carbohydrates - a mechanism for the sink regulation of photosynthesis. Plant Journal 3, 817-828. Lam, E., Green, P. J., Wong, M. and Chua, N-H. (1989). Phytochrome activation of two nuclear genes requires cytoplasmic protein synthesis. EMBO Journal 8, 2777-2783. Lamb, C. J. and Dixon, R. A. (1977) The oxidative burst in plant disease resistance. Annual Review of Plant Physiology and Plant Molecular Biology 48, 25 1-275. Leung, J., Merlot, S. and Giraudat. J. (1997). The Arabidopsis ABSCISIC ACIDINSENSITIVE2 (AB12) and ABIl genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction. Plant Cell 9, 759-771. Levine, A., Tenhaken, R., Dixon, R. A. and Lamb, C. J. (1994). H202 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79, 58 3-5 93. Li. H-M., Altschmied, L. and Chory, J. (1994). Arabidopsis mutants define downstream branches in the phototransduction pathway. Genes and Development 8, 339-349. Li, H-M., Culligan, K., Dixon, R. A. and Chory, J. (1995). CUE]: a mesophyll cell-specific positive regulator of light-controlled gene expression in Arabidopsis. Plant Cell 7 , 1599-1 610. Lozoya, E., Block, A., Lois, R., Hahlbrock, K. and Scheel, D. (1991). Transcriptional repression of light-induced flavonoid biosynthesis by elicitor treatment of cultured parsley cells. Plan? Journal 1, 227-234. Mason, H. S., DeWald, D. B., Creelman, R. A. and Mullet, J. E. (1992). Coregulation of soybean vegetative storage protein gene expression by methyl jasmonate and soluble sugars. Plant Physiology 98, 859-867. Merlot, S. and Giraudat, J. (1997). Genetic analysis of abscisic acid signal transduction. Plant Physiology 114, 75 1-757. Millar, A. J., McGrath, R. B. and Chua, N-H. (1994). Phytochrome phototransduction pathways. Annual Review of Genetics 28, 325-349. Millar, A. J., CarrC, I. A., Strayer, C. A., Chua, N-H. and Kay, S. A. (1995). Circadian clock mutants in Arabidopsis identified by luciferase imaging. Science 267, 1161-1 163. Mistra, S., Muller, A. J., Weiland-Heifecker, U. and Jurgens, G. (1994). Thefusca genes of Arabidopsis: negative regulators of light responses. Molecular and General Genetics 244, 242-252.

SIGNAL TRANSDUCTION NETWORKS

73

Mohr, H. ( 1994) Coaction between pigment systems. In “Photomorphogenesis in Plants” (R. E. Kendrick and G. H. M. Kronenberg, eds) pp. 353-373. Kluwer Academic Publishers, Dordrecht, The Netherlands. Mol, J., Jenkins, G. I., Schafer, E. and Weiss. D. (1996). Signal perception, transduction, and gene expression involved in anthocyanin biosynthesis. Critical Reviews in Plurit Science 15, 525-557. Monroy, A. F. and Dhindsa, R. S . (1995). Low-temperature signal transduction: induction of cold acclimation-specific genes of alfalfa by calcium at 25°C. Plunr Cell 17, 321 -33 1. Muranaka, T., Banno, H. and Machida, Y.(1994). Characterization of tobacco protein kinase NPKS, a homolog of Saccharomyces cerevisiae SNFl that constitutively activates expression of the glucose-repressible SUC2 gene for a secreted invertase of S. cerevisiae. Molecular and Cellular Biology 14, 2958-2965. Neuhaus, G., Bowler, C., Kern, R. and Chua. N-H. (1993). Calciudcalmodulin-dependent and -independent phytochrome signal transduction pathways. Cell 73, 937-952. Nordin, K., Heino, P. and Palva, E. T. (1991). Separate signal pathways regulate the expression of a low-temperature-induced gene in Arahidopsis thaliana (L.) Heynh. Plant Molecular Biology 16, 1061-1071. Shacklock, P. S., Read, N. D. and Trewavas, A. J. (1992). Cytosolic free calcium mediates red light-induced morphogenesis. Nature 358, 753-755. Sheen, J. (1990). Metabolic repression of transcription in higher pl,ants. Plunt Cell 12, 1027-1 038. Shirasu, K., Nakajima, H., Rajasekhar, V. K., Dixon, R. A. and Lamb, C. J. (1997). Salicylic acid potentiates an agonist-dependent gain control that amplifies pathogen signals in the activation of defense mechanisms. Plant Cell 9, 261-270. Singh, N. K., Bracker, C. A., Hasegawa, P. M., Handa, A. K., Buckel, S., Hermondson, M. A,, Pfankoch, E., Regnirr, F. E. and Bressan, R. A. (1987). Characterization of osmotin. Plant Physiology 85, 529-536. Smeekens, S. and Rook, E (1997). Sugar sensing and sugar-mediated signal transduction in plants. Planf Physiology 115, 7-1 3. Stapleton, A. E. (1992). Ultraviolet radiation and plants: burning questions. Plant Cell 4, 1353-1 358. Terzaghi, W. B., Bertekap, R. L. and Cashmore, A. R. (1997). Intracellular localization of GBF proteins and blue light-induced import of GBF2 fusion proteins into the nucleus of cultured Arahidopsis and soybean cells. Plant Journal 11, 967-982. Unvin, N. A. R. and Jenkins, G . I. (1997). A sucrose repression element in the Phaseolus vulgaris rbcS2 gene promoter resembles elements responsible for sugar stimulation of plant and mammalian genes. Plant Molecular Biology 35, 929-942. van der Meer, I. M., Stuitje, A. R. and Mol, J. N. M. (1993). Regulation of general phenylpropanoid and flavonoid gene expression. In “Control of Plant Gene Expression” (D. P. s. Verma, ed.) pp. 125-155. CRC Press, Boca Raton, Florida. Vidal, S., Ponce de L e h , I., Denecke, J. and Palva, T. E. (1997). Salicylic acid and the plant pathogen Envinia carotovora induce defense genes via antagonistic pathways. Plant Journal 11, 115-123. von Schaewen, A., Stitt, M., Schmidt, R., Sonnewald, U. and Wilmitzer, L. (1990). Expression of yeast-derived invertase in the cell wall of tobacco and Arahidopsis plants leads to inhibition of sucrose export, accumulation of carbohydrate and inhibition of photosynthesis, and strongly influences the growth and habitus of transgenic tobacco plants. EMBO Journal 9. 3033-3044. Xu, Y., Chang, P-F., Liu, D., Narasimhan, M. L.. Raghothama, K. G., Hasegawa, P. M. and Bressan, R. A. ( 1994). Plant defense genes are synergistically induced by ethylene and methyl jasmonate. Plant Cell 16, 1077-1085.

This Page Intentionally Left Blank

Plate 1 . Light micrographs of abaxial epidermis, stained with silver-rubeanate, of (A) Centuureu scubiusu and (B) Leonrodon hispidus grown with 15 mol rn-? calcium. The black deposits indicate the presence of calcium. The scale bar represents 100 pm. (from De Silva et al., 1996).

Plate. 2. Secondary ion mass spectrometry images of Ca and Mg isotopes in a corn root rip. Treatment was as described in the text with a 30 min labelling period and a 2 min ice-cold rinse to remove superficial and apoplastic label. The four isotope maps (tracers in right hand panels) were collected in a CAMECA IMS 4f operated in ion microprobe mode, utilizing direct detection on the electron multiplier and Faraday cup (Lazof et al., 1996a). The brighter the image the greater is the mass signal intensity. The labelled plant specimens were excised from the plant, quench-frozen, cryosectioned (1 0 p m thickness) and slowly freeze-dried. Employing the ‘depletion’ pre-treatment and these operating conditions, all of the 44Ca2+and 26Mg2+represents label which has arrived in the root tip during the labelling period. The three arrows point to the edge of the root, and the asterisk indicates an area where the section has split during freeze-drying. Bar = 80 pm.

Mechanisms of Na+ Uptake by Plant Cells

ANNA AMTMANN and DALE SANDERS

The Plant Luboratoiy. Biology Department. PO Box 373. University of York. York YO1 5yW; UK

1. Introduction

.......................................................................................................

A . Salinity Toxicity and Salinity Tolerance ................................................... B . Exclusion, Uptake and Sequestration of Na+ ...........................................

76 76 17

I1 . Electrochemical Potential Differences for Na’ Across the Plasma and

........................................................................................

78

111. Carrier-Mediated Entry of Na’ .........................................................................

80

IV. Channel-Mediated Entry of Na’ ..................................................... A . Ionic Selectivity of Ion Channels . B . Inward-Rectifying Channels ............................ C . Outward-Rectifying Channels ................................................................... ......................................... D . Voltage-Independent Channels ......... E . Co-residency of Different Channel Types ..............................

82 82 84 88 88 89

Vacuolar Membranes

V. Contributions of Different Channel Types to Na+ Entry in Physiological Conditions ......................................................................................................... A . Semi-quantitative Dissection of Fluxes .................................................... B . Relative Activity of Different Channel Types Determines Rate of Na+ Uptake .......................................................................................... VI

.

Regulation of Monovalent Cation Influx Across the Plasma Membrane ......... A . Voltage ................... ........................................................ B. External Ca’* and p ......................................................... C . Cytosolic Ca” and pH ......................................... D . External and Cytosolic Na’ . .................................................... E. ATP ....................................................................... F. Other Regulators ...............................................................................

VII . Comparison of Salt-Sensitive and Salt-Tolerant Genotypes or Cell Lines ....

91 91 95 96 97 97 101 101 102 102

103

Advaices in Buimical Research Vul . 29

incorporating Advances in Plant Pathology ISBN 11-12-00592’3-0

AII righla

Copyright 0 1999 Academic Press reproduction in any form rwerved

76 VIII.

A. AMTMANN and D. SANDERS

Future Work .................................................................................................... Acknowledgements ......................................................................................... References .......................................................................................................

103 104

I04

Soil salinity affects vast areas of land globally, with a particularly high impacr in some agricultural intensively used soils due to irrigation practice. A diverse range of plants is able to thrive on saline soils but all major crop species are intolerant to salt. Identijcation of pathways for Na+ transport across plant cell membranes has been highlighted as comprising a key gap in our understanding of salt tolerance in plants. During the last few years there have, however; been remarkable advances in this area as Na+ permeable ion channels in plant cells have been characterized. This review summarizes the present knowledge regarding Na' transport pathways across plant membranes. In particular; data on selectivity, conductance, abundance and regulation of the major cation uptake channel types have been collected an,d this information has been integrated into a simple model in order to address the following questions: (i) how much Nu+ enters the cell through an ensemble of different channel types in saline conditions? (ii) what is the relative contribution of each channel type to the total Na' inward current? (iii) how does modulation of the activity of the different channel types affect the ability of the plasma membrane to discriminate between K+ and Na' ? The model calculations underline the importance of voltuge-independent non-selective cation channels in Nu+-uptake and suggest that future reseurch in the field of salt tolerance in plants should include studies on the regulation oj' this channel type.

I. INTRODUCTION A. SALINITY TOXICITY AND SALINITY TOLERANCE

Soil salinity has a major impact on plant growth and affects about 6% of the total global land area (Flowers and Yeo, 1995). Increasingly, intensive irrigation practices are resulting in secondary salinization of agricultural soils, such that it has been estimated that 10 X lo6 ha per nnnum of irrigated land are abandoned due to salinization and alkalization (Szabolcs, 1987). Since crop productivity of irrigated land in many areas is much higher than that of non-imgated land the coincidence of irrigation and salinization threatens current agricultural productivity (Flowers and Yeo, 1995). Although all major crop species are intolerant of high levels of salinity, a taxonomically diverse range of plant species are able to grow and thrive on saline soils. In extreme cases, where growth is actually enhanced by the presence of NaCl, such species are said to be halophytic, although the spectrum between extreme halophytes and extreme glycophytes is a continuous one which embraces varying levels of salt tolerance. Studies of the mechanisms that underlie salt toxicity and salt tolerance of plants have revealed the involvement of many aspects of cellular, tissue and whole plant biology (for reviews see Rains, 1972; Flowers et al., 1977; Greenway and Munns, 1980; Munns el al., 1983; Cheeseman, 1988; Gorham, 1992; Munns, 1993; Niu et al., 1995; Serrano, 1996; Yeo, 1998). The two principal adverse effects of salinity

MECHANISMS OF Na' UPTAKE BY PLANT CELLS

77

in non-tolerant plants are osmotic stress and toxicity of Na' andor C1- (Serrano, 1996), whereas ion deficiencies (particularly of K+ and Ca2+;Lauchli et al., 1994), decrease of COz fixation and inhibition of protein synthesis probably follow as secondary effects (Marschner, 1995). Salt tolerance involves tissue- and whole plant integration of many different transport processes, as well as compartmenfation of ions and de n o w synthesis of organic osmolytes at the cellular level. In a seminal and critical review of processes limiting growth on saline soils, Munns (1993) points to a lack of research on the control of Na+ and CI- transport across the plasma and vacuolar membranes and concludes that 'advances in salt tolerance at the molecular level will lie in manipulating the expression and structure of proteins that control transport of salt across membranes'. Identification of pathways for plasma membrane Na' transport in plants has more recently been highlighted as comprising a key gap in our understanding of ionic homeostasis during saline stress (Niu el al., 1995). During the last two years there have, however, been some remarkable and significant advances emanating from research on Na' uptake mechanisms by plant cells, and this comprises the topic of the present review. B. EXCLUSION, UPTAKE AND SEQUESTRATION OF Na'

Plant cells in general, and halophytes in particular, face a dilemma in the context of Na' uptake from the soil. On one hand the absorption of Na+ is desirable as a metabolically cheap way of generating high internal osmotic pressure in response to high external osmotic pressure, thereby lowering cell water potential and sustaining turgor. On the other hand, Na' is cytotoxic at cytosolic concentrations in excess of about 100 mM. These cytotoxic effects of Na' are dual. First, the high charge:mass ratio (in comparison with K ' ) disrupts water structure and lowers hydrophobic interactions within proteins, thus reinforcing the overall destabilizing effect of high ionic strength on protein structure through decrease in hydrostatic forces within proteins (Pollard and Wyn Jones, 1979; Wyn Jones and Pollard, 1983). Second, Na+ can inhibit enzyme function more specifically, either directly by binding to inhibitory sites or indirectly by displacing K' from activation sites (Serrano, 1996). In both instances, competition between Na' and Kf is likely to be critical, and therefore the Na+:K' ratio in the cytosol is likely to be a more critical factor in determining Na' toxicity than the cytosolic Na' concentration per se. Two solutions to the dilemma of Na+ absorption are evident. A simple one, probably operational both in moderately halotolerant and halophytic species, is the efficient exclusion of Nab from the plant (Munns, 1985; Schubert and Lauchli, 1990). In addition, since halophytes exhibit a marked propensity for Na' accumulation, that Na+ which does enter the cell must be efficiently sequestered in the vacuolar lumen to prevent cytotoxicity. It is clear, then, that the regulation of Na* transport across the plasma and vacuolar membranes will comprise a critical factor in determining the specific manner in which plant cells handle extracellular Na' loads.

78

A. AMTMANN and D. SANDERS

11. ELECTROCHEMICAL POTENTIAL DIFFERENCES FOR Na’ ACROSS THE PLASMA AND VACUOLAR MEMBRANES Evaluation of driving forces is a central element in understanding transport processes. The driving force for transport of an ion across a membrane has two components, a chemical component established by the concentration difference between the cytoplasm and the extracytosolic compartment, and an electrical component consisting in the electrical potential difference between the two compartments. Quantitatively, the driving force or electrochemical potential difference can be described as

ASNa= zFV, + RT In

( [Na+l,,,IINafl,,,},

where V,, is the electrical potential difference across the membrane referenced to the extracytosolic side of the membrane, “a’] is the activity (“active” concentration depending on the ionic strength of the medium) of Na+, the subscripts cyl and e x t refer to the cytosolic and extracytosolic sides respectively and z, F, R and T have their usual meanings. At the plasma membrane, the chemical driving force for Na’ will obviously vary depending on the extent of salinity. At low salinity (<100mM Na’ in the soil), it is probably safe to assume that [Na’]],,, is close to [Nat],,,, since X-ray microanalytical studies have demonstrated that [Na+Icytdoes not exceed I50 mM in living cells (Binzel et al., 1988; Hajibagheri and Flowers, 1993; Hajibagheri ef al., 1987). At moderate to high salinity, the chemical driving force will become progressively directed inward, and in cells able to withstand high salinity, the ratio of Na’ concentrations across the plasma membrane might well exceed fivefold. The situation can be exacerbated in the shoot, where transpirational deposition of Na’ leads in some species to its accumulation in the apoplast. However, even a 10-fold gradient is equivalent in energetic terms (see Eq. I ) to only -5.7 kJ/mol. By contrast V, normally attains a value of - 120 mV to -200 mV, equivalent to a driving force on Na’ of between -11 and -19kJ/mol. Some studies have suggested that the membrane depolarizes in response to addition of NaCl to the external medium (Cakirlar and Bowling, 1981; Katsuhara and Tazawa, 1990; Kourie and Findlay, 1990), thereby reducing the driving force provided by V,. Even so, negative values of V , always prevail. Overall, we can safely conclude that there will invariably be a cytosol-directed driving force on Na’ across the plasma membrane amounting to between - 10 and -20 kJ/mol, with the electrical term in Eq. 1 dominating. Sodium entry into plant cells is therefore passive, while its export across the plasma membrane must be energized. The mechanism of energization of Na’ efflux remains to be established in higher plants. ? b o obvious possibilities are that transport is driven by an ATPase, as is likely in Succharomyces cerevisiue (Haro et al., 1991), or that Na+ efflux is coupled to, and hence driven by, passive H+ influx, as is likely in Schizosuccharomyces pornbe (Jia et al., 1992). Evidence for the existence of the latter pathway in the

MECHANISMS OF Na' UPTAKE BY PLANT CELLS

79

plasma membrane of higher plants has derived from experiments showing stimulation of whole-tissue Na' efflux by low external pH (Colombo et al., 1979; Jacoby and Teomy, 1988; Mennen et al., 1990) and for the halophyte Atriplex, a plasma membrane Na+/H antiporter has been proposed on the basis of membrane vesicle studies (Braun et al., 1988). Na+-ATPases on the other hand have been found in the plasma membrane of marine algae (Wada et al., 1989; Pick, 1992) but so far evidence for Na'-pumps in higher plants is lacking. Although reasonably large, the electrochemical potential difference which drives Naf into the cell across the plasma membrane is by no means an insuperable obstacle for energized ion efflux. For example, all plant cells are competent to grow at an external Ca'' concentration of 1 mM while maintaining cytosolic Ca2+ in the region of 200nM. Together with a V,,, of -150mV, this large concentration difference generates an electrochemical potential difference for Ca2' across the plasma membrane of -50kJ/mol, which is at least twofold greater in energetic terms than that for Na'. Problems with high external Na' loads must, then, relate to kinetics more than thermodynamics. In other words, the inherent permeability of the plasma membrane to Na' will be high enough to admit sufficient Na' to the cytosol to cause toxicity in cases where plants are intolerant to high apoplastic Na' . Exploration of the identities of these Na t-permeable pathways comprises the principal topic of this review. A cytosol-directed electrochemical potential difference for Na' is also normally present across the vacuolar membrane. Such measurements as have been made (all on NaC1-stressed tissue) suggest that Naf is usually two- to eightfold higher in the vacuolar lumen (Hajibagheri er al., 1987; Binzel et al., 1988), whereas V,,, is cytosol-negative and probably of the order -20mV (Allen and Sanders, 1997). This yields an overall driving force into the cytosol of around -6 kJ/mol. In accord with this finding, Na' export from the cytosol (i.e. sequestration into the vacuolar lumen) is powered by exchange with H t ions which move passively from the highly acidic lumen (see Blumwald and Gelli (1997) for review). A role for this transport system in salt tolerance appears likely from the observations that in some species, including barley and Mesembryanthemum crystallinum, activity is enhanced after exposure of the parent tissue to NaCl (Garbarino and Dupont, 1988; Barkla et al., 1995). Fig. 1 summarizes this discussion on the principal energized transport systems moving Na' uphill across the vacuolar and plasma membranes. The Na+/H+ exchange is shown at the vacuolar membrane, powered ultimately by a H'pumping ATPase which generates the cytosol-directed electrochemical potential difference for H + across this membrane. At the plasma membrane, the joint possibilities of a Na+-pumping ATPase and an N+/H' exchanger are both shown: neither transport system has been firmly identified. In the latter case, Na' export would be driven indirectly, as it is at the vacuolar membrane, by an H'-pumping ATPase. Fig. 1 also includes a number of additional transport systems which provide the context for the remainder of this review. A Na':K+ carrier at the plasma membrane

80

A. AMTMANN and D. SANDERS

nH+ L

Na+ . _ ,-. . .\

Fig. I . Schematic view of possible pathways for Na' membranes.

transport across plant

might serve both as a Na' leak, and to drive energized Kf uptake (section 111). Three classes of cation-selective channel are also depicted: voltage-dependent systems which respectively catalyse either the inward or the outward flow of K + (and which to varying extents allow passage of Nat) and a voltage-insensitive channel which fails to discriminate markedly between Na+ and K ' . The properties of these channels and their integrated actions are discussed in detail in Sections IV, V and VI. It should be mentioned that transport of Na' across the plasma membrane via exo- and endocytosis (vesicular shuttle) has been suggested for the marine alga Acetabuluriu (Mummert and Gradmann, 199 1; Amtmann and Gradmann, 1992). The existence of a vesicular compartment within the cytoplasm involved in ion transport has also been postulated for Chaetomorpha (Dodd et al., 1966), Atripla (Liittge and Osmond, 1970) and Sperguluriu (Lazof and Cheeseman, 1986).

111. CARRIER-MEDIATED ENTRY OF Na' Carrier-type transport systems are distinctive in exhibiting conformational changes in the transport protein as part of the transport mechanism. These conformational changes reflect themselves as an apparent reorientation of transport binding sites from one side of the membrane to the other and back again during transport (Stein, 1990). The conformational changes endow carriers with the potential for ion coupling, where binding of a specific ion to one transport binding site facilitates '

MECHANISMS OF Na' UPTAKE BY PLANT CELLS

81

binding of a solute or ion at a discrete site, thereby provoking transport of the second solute or ion. The process of ion-coupled transport is referred to as symport when transport of the two ligands is in the same direction, and antiport when transport is in the opposite direction. The dominant ion pump at the plasma membrane of higher plants is a H+-pumping ATPase which sets up an inwardly directed electrochemical potential difference for H across the plasma membrane. Accordingly, the majority of solute transport systems at the plasma membrane is energized by passive inward flow of H+. Substrates which are accumulated via carriers mediating H t -symport include sugars and amino acids (Bush, 1993), C1- (Felle, 1994), P, (Ullrich-Eberius et al., 1984), SO,'- (Lass and Ullrich-Eberius, 1984) and K ' (Maathuis and Sanders, 1994). In addition to this range of H+ symport systems, there is increasing evidence that Na'-symport systems play a role in solute absorption. In the freshwater charophyte algae Churu and Nitella, Na' -coupling has been found for uptake of K t , urea and lysine (Smith and Walker, 1989; Walker and Sanders, 1991; Walker et al., 1993). Among higher plants, symport of Na' with K+ has been reported in some freshwater species (Elodea, Wlisneria), but not in any of the terrestrial species (including barley, wheat and Ambidopsis) in which it was looked for (Maathuis et ul., 1996). A carrier-type transporter - HKTl - cloned from a cDNA library of K'-starved wheat confers high affinity Na'-coupled K' transport when expressed in yeast or in Xenopus oocytes (Rubio et al., 1995). Furthermore, point mutations in HKTl which increase K+/Na+ selectivity can confer salt-tolerance in yeast. Although the physiological role of this carrier in K+ uptake remains controversial (Rubio et al., 1996; Walker et al., 1996), the possibility that this transporter represents a major pathway for Na' uptake into the roots of terrestrial plants deserves consideration. With both Na+ and K+ present at low levels, HKTl exhibits a high transport affinity for the two ions, which are transported with micromolar K,,s in yeast (Rubio et al., 1995). However, as the [Na'],,, is increased into the millimolar range, Na' appears to compete successfully for the K+ binding site, simplifying the transport reaction to one of low affinity Na' uptake only while blocking K+ uptake (Cassmann ct al., 1996). If retained in planta, this property would confer on HKTl the properties of a transport system responsible for low affinity uptake of N a ' . Circumstantial evidence for HKTl -mediated low affinity Na uptake is available from sosl mutants of Arabidopsis (Wu et al., 1996). sosl mutants are hypersensitive to NaCl - about 20 times more so than wild type. The mutants are also defective specifically in high affinity K ' uptake (i.e. from solutions containing 1 mM or less K+) and in low affinity Na+ uptake (Wu er al., 1996; Ding and Zhu, 1997). Besides the apparent linkage of K ' and Na' fluxes, a further observation might be taken to suggest the involvement of HKTl: the inhibition of low affinity Naf uptake is apparent only in tissue which has been K' starved (Ding and Zhu, 1997) and this coincides with the finding that HKTl expression is derepressed by K+ starvation (Gassmann et al., 1996). However, evidence that HKTl mediates significant transport at the plasma

82

A. AMTMANN and D. SANDERS

membrane of root cells is currently lacking. Early studies with radiotracers have revealed no effect of Na+, neither in micromolar nor in millimolar concentrations, on high-affinity K+ uptake into barley roots (Epstein, 1961; Epstein et a/., 1963) More recent electrophysiological studies on the roots of terrestrial plants have also failed to reveal the type of synergism at micromolar [Na+Iextand [Kflex,which would also be predicted were HKTl a major route for Naf and K+ absorption, although evidence has been obtained for high affinity, Na+-independent uptake of K+ (Walker et al., 1996; Maathuis et al., 1996). Furthermore, even after K' starvation, Na' influx in sosl mutants is only reduced by 32% (Ding and Zhu, 1997), suggesting that, even if HKTl is involved in mediating transport, this carrier accounts for only a minor fraction of total uptake. In summary, in the absence of definitive information on the properties of HKTl in plunta (such as might be obtained from T-DNA insertional mutants, Krysan el al., 1996), it is too early to state whether this transport system contributes significantly to Na+ uptake across the plasma membrane. The balance of the evidence to date suggests that, if there is a contribution, it is a relatively small one. Another gene from wheat, LCT1, was also found to complement yeast mutants deficient in K+ uptake (Schachtman et al., 1997). Yeast mutants expressing LCTl were able to take up Rb+ and Naf with low affinity and Na' uptake was reduced by addition of Ca2+.LCTl does not resemble any other known gene of ion transporters. Hydrophobicity analysis predicts the existence of 8-10 transmembrane spanning helices, a number which points to LCTl being a carrier rather than a channel. Northern blots show low-abundance expression of LCTl mRNA in wheat leaves and roots. The physiological role of LCTl in plantu remains to be revealed.

IV. CHANNEL-MEDIATED ENTRY OF Na+ A. IONIC SELECTIVITY OF ION CHANNELS

Ion channels are distinguished from carriers by their capacity to catalyse transmembrane ion fluxes without attendant conformational changes in the transporter. This property endows channels with high turnover rates - of the order lo6 to lo8s-' (Hille, 1992). Nevertheless, channels undergo conformational changes between open and closed states in the regulation of ion fluxes; this property is known as gating. Channel gating can be controlled by membrane voltage, by reversible binding of ligands or by covalent modification, for example through phosphorylation. Ion channels are most commonly studied with the patch clamp technique (Hamill er al., 1981; Hedrich and Schroeder, 1989), although they can also be studied in synthetic planar lipid bilayers after incorporation of purified membranes (Miller, 1986; White and Tester, 1992). In both cases, the ionic fluxes through the channels are measured as electrical currents, and control of solution composition is achieved

MECHANISMS OF Na' UPTAKE BY PLANT CELLS

83

specifically on each side of the membrane. A major experimental task is to identify which ions are responsible for carrying the current through the channel, a property which will be defined by the ionic selectivity of the channel. In the present case, we are, of course, interested in ascertaining the extent of Na+ permeation through ion channels. The most common approach to identifying the ionic components of the channel-mediated current involves measurement of its reversal potential, Ere, - the zero-current potential at which net ion flow through the channel changes from the inward to the outward direction as the membrane potential becomes more positive. The resultant values of E,, in different solutions are then compared with the equilibrium potentials of specific ions, E,,,. E,,, is the voltage at which the chemical and electrical driving forces for a given ionic species are equal in size but opposite in sign (Ajilon= 0 in Eq. 1). If a channel was 100% selective for K', Ere, would be equal to EK. However, no channel is perfectly selective and therefore Ere, lies somewhere between all relevant Elonswhen measured in solutions with more than one permeant ion present. In most studies, the Goldman-Hodgkin-Katz (GHK) equation (see for example Hille, 1992) is then applied to determine relative ionic permeabilities of the channel, usually expressed with respect to K+ ions. The GHK equation has been formulated for a partitioning electrodiffusion model which makes two basic assumptions: that (a) the ions move independently through the channel and that (b) the electrical field across the membrane is constant (Hille, 1992). In other words, there are no ion-ion interactions and no ion-protein interactions. Generally, neither of these assumptions is correct for plant ion channels. For example, E,,s have been shown to be dependent not just on the concentration difference across the membrane, but on concentration per se, which implies ion-ion interaction (White and Ridout, 1995); permeability sequences obtained from Ere$ do not match permeability sequences obtained from relative currents, and this implies ion-protein interaction (Elzenga and Van Volkenburgh, 1994; VCry et al., 1995; Amtmann et al., 1997); E,,s can depend on the side of the membrane which is exposed to Na', or K + , again implying ion-protein interaction (Schroeder et al., 1984; Schauf and Wilson, 1987b). Thus, E,.s may reflect the affinity of the channel protein for a certain ion rather than permeation of this ion through the pore. Affinity can be a poor guide to permeation, since tight binding (high affinity) can impede passage of the ion through the channel (Johannes and Sanders, 1995). As an alternative to Ere, measurement, ionic fluxes through channels can be estimated simply from the steady-state currents in single-salt solutions, or from the increment in current when one salt is added to another. However, if ionic species are not transported or selected for independently, these experiments will also fail to report accurately how much of an ionic species is transported in mixed solutions. Attempts have been made to characterize ion permeation in mixed solutions by using refined experimental procedures and alternative models (Johannes and Sanders, 1995; White and Ridout, 1995; Gradmann, 1996; Gradmann et al., 1997; Allen et al., 1998) but none of these approaches is sufficiently simple or independently validated to be used in a general assessment of channel selectivity.

84

A. AMTMANN and D. SANDERS

Nevertheless, these drawbacks and uncertainties relate to a refined analysis of ion permeation through channels. In order to derive a semiquantitative picture of Na+-permeation through ion channels, we have for a wide variety of monovalent cation channels collated estimates of relative permeabilities of Na+ with respect to Kf (PNa:PK) derived either through measurement of E,,s or of relative currents (or conductances). The collated data are shown in Table 1, and where comparisons can be made, fairly consistent estimates result. We have classified the ion channels according to their gating properties, and we now consider in turn the properties of each of the three major classes with respect to Naf permeation and its control. B. INWARD-RECTIFYING CHANNELS (IRCS)

As their name implies, inward-rectifying channels pass current into the cell, but not out of it. In this sense they behave like valves, opening in pure K+ solutions when voltage becomes more negative than EK. The opening response is also timedependent, with the channels opening over a period of tens or even hundreds of milliseconds after a permissive voltage pulse is applied (Schroeder et af., 1987; Colombo and Cerana, 1991). The principal function of inward-rectifying cation channels is thought to be K+ uptake (Maathuis et al., 1997). IRCs have been described for many different plant cell types and the Arubidopsis IRCs KATl and AKTl were the first plant channels to be identified at a molecular level (Anderson et al., 1992; Sentenac et al., 1992). Both KATl and AKTl belong to the so-called Shaker class of ion channel, with each subunit possessing six transmembrane spans, plus a pore region which defines ionic selectivity (Becker et al., 1996; Uozumi et al. 1995). Like other Shaker K+ channels, KATl and AKTl function as tetramers (Daram et al., 1997). Whereas KATl is dominantly expressed in guard cells (Nakamura et al., 1995), AKTl is preferentially expressed in the peripheral cell layers of mature roots (Lagarde et al., 1996). Inward rectifiers are generally very selective for K+ over Na+. The lowest PNa:PKratios have been reported for KATl (VBry et ul., 1995) and KSTl (a similar channel cloned from potato, Miiller-Rober et al., 1995) when expressed in Xenopus oocytes. This corresponds to low values of PNa:PKdetermined for native inward-rectifying currents in some plant protoplasts (e.g. Gassmann and Schroeder, 1994). However, it has to be pointed out that there are inward-rectifying channels with much higher Na' permeability. Tyerman et al. (1997) found that wheat root cortex protoplasts display three types of hyperpolarization-activated whole-cell inward currents. One of them has a 'spiky' appearance and is carried by K+ as well as Na*, the second one shows typical IRC features including high selectivity for K+ over Na+, whereas the third one (so-called S(1ow)Na) mediates considerable movement of Naf. IRC and SNa currents have very similar activation kinetics but could clearly be distinguished by the fact that SNa was only observed in protoplasts that did not show IRC activity in high-Kf solutions. Similarly, Amtmann et al. (1997) reached the conclusion that inward-rectifying channels with similar gating kinetics but different Na' :K' permeability contribute to the whole-cell time-

TABLE I Na':K+-penneubiliry of plant p l a s m membrane ion channels

Specie5

Cell type

Recording mode

_ __-

.

PN,

Method

:PK

~~~~

Ion concentration (d) (cytoplasmid external)

Reference

.

~~

Inward rectifying channels . _

.-.

-

Whole cell"'

f?.eosrJ

-

External 100 K Wry er al. (1995) l00Na 115K-115Na Uozumieral. (1995) 120 W25 K + 95 Na Marten er al. (1996)

KAT I

Whole cell

KAT 1

Whole cell

0.05

Relative I"'

175 Wl50 Na

Bertl et al. (1995)

AKTl

Whole cell

0.05

Relative

I75 W135 Na

Bertl

KATl

Whole cell"'

0.07

Relative

KST I

Whole cell'"

<0.01

ER"

Root hair cells Leaf mesophyll Root cortex Aleurone Coleoptile cortex Guard cells Guard cells

Whole cell Whole cell Whole cell Whole cell Whole cell Whole cell'" Whole cell

0.01 0.02 0.02 0.03 <0.03 <0.02 0.06

Relative I"'

Arahidopsis thaliana (Xenopus) Arabidopsis fhaliana fSfP) Arabidopsis rhaliana

~~~

0.002 0.2 0.02 very low

KAT I

Em".

relative G'" Relative G"' Relative I

el

a/. (1997)

Arabidopsis thaliana (yeast)

Arabidopsis ~Iialianu (Xenopus) Solanrrm ruberosum (XenopusJ Trrticum aestiwm A l m a sarii'a Zea mays Hordeum wlgore zpu my'" Vicru,faba

Kcia fabo

F5'

Ere,

Relative I"' E,\

Em, Ere\

Ere\

External 1 15 K * 115 Na n.d. IOOWlOONa 100WIONa 100W30Na 100W100Na 100 W30 Na 100W100Na 105 W101 Na

Schachtman er a/. (1992) MUller-Rober er al. (1995)

Gassmann and Schroeder (1994) Kourie and Goldsmith (1992) Roberts and Tester ( I997b) Bush et al. (1988) Hedrich er a / . (1995b) Blatt and Gradmann (1997) Schroeder et a / . (1987)

TABLE I Contd.

Species Hordeum vulgare Trianea bogorensis Arabidopsis fhaliana Hordeum vulgare

Cell t w Culture cells (scutellum) Root hair Root conex Root xylem parenchyma

Ion concentmion (d) (cytoplasmid external)

Recording mode Whole cell Whole cell"' Single channel Whole cell

0.07 0.17 0.15 0.17 0.2

Em", relative P' Relative f9' Ere" Em,

100 W100 Na

-

Reference

__

Amtmann e f al. (1997)

External 5 K 5 Na Grahov (1990) 10 W10 Na Maathuis and Sanders (1995) Wegner and Raschke (1994) 120 W30 Na

Outward-rectifying channels

Arabidapsis fhaliana Nicotiana tabarum

Xylem parenchyma (KORC) Leaf mesophyll Culture cells

Triticwn sp

Root

Samanea suman Kcia faba

F'ulvinar motor cells Guard cell

Wcia fnba Nitellopsis obtusa Asclepias ruberosa

Guard cell Nodal cells Suspension culture cells (callus) Suspension culture cells Root hair Steele KCOl Cotyledons Young leaf cells

Hordeum vulgare

Nicotiana tabacum Tnanea bogorensis .&a mays Araidopsis thaliana (SfpJ Amaranthus rncolor Zusfera muellen

Single channel

Very low

120 W30 Na

Single channel Whole cell

Very low

Whole cell, Single channel Whole cell Whole cell, Single channel Whole cell Single channel Whole cell, Single channel Whole cell

0.03

250 Nd50K 103 K + 5 N a /5 K + 200Na 100 Wl00Na

0.05 0.05

I39 W6 K + 25 Na 100W100Na

Moran er al. (1990) Schauf and Wdson (1987a)

0.13 0.07 0.07

105 W101 Na 50 Nd50 K 100 Nd100 K 100WIWNa 1 0 0 K + 5 Na /10 K + 0 50 Na External 5 K- 5 Na 100WlOONa 30 W30 Na IOOK/3K+lOONa 100 W100 Na

Schroeder ef al. (1987) Azimov er al. (1987) Schauf and Wilson (1987h)

Whole cell"' Single channel Whole cell Single channel Single channel

0.01

0.12 0.09 0.08

0.09 0.16 0.16 0.2

-

Wegner and De Boer (1997b) Spalding et d.(1992) Murata et al. (1994) Schachtman et a/. (1991)

Van Duijn e f al. (1993) Grahov (1990) Roberts and Tester (1997b) Czempinski et al. (1997) Terry e r a / . (1991) Garrill et a/. ( 1994)

Arabidopsis rhaliana Arabidopsis rhaliana Lilium longiflorum Arabidopyis thaliana Hordeum vulgare Cliva haemanrhus

Root conex Suspension culture cells Pollen grains Leaf mesophyll Xylem parenchyma (NORC) Endosperm

10 WIO Na 100lUIONa

Maathuis and Sanders (1995) Cerana and Colombo (1992)

Ere\

External 2 K 2 Na 250 Nd50 K 120 W30 Na

Obermeyer and Blatt (1995) Spalding er 01 (1992) Wegner and Raschke (1994)

Exv.

100 Nd25 K

Stoeckel and Takeda (1989)

Single channel Whole cell Whole cell"' Single channel Whole cell

0 45 I I

Single channel

I

EEm"

-

relative G -

-_

~

Voltage-independent channels -

Secale cereale

Root

Single channel""'

100 WlOO Na 100NdlOONa

White and Tester (1992)

0.6

Single channel"" Single channel"" Whole cell

0.37 0.84 0.78 0.62 0.6

1 W1 Na loo0 WIoo0 Na 100WlM)Na 100 Nd100 Na 10 K + 4 Nd100Na

White and Ridout (1995)

0.73

Secale cereale

Root

Secale cereale

Root

Triticum aestivum

Root conex

Hordeum d g ~ n

Single channel

1

Zea mays Pisum safivum

Culture cells (scutellum) Root cortex Leaf epidermis

Single channel Single channel

Secale cereule

Root epidermis

Whole cell

2. I 5.1 1.7 3.8 9.9

Em. EX,

Tyerman et al. (1997)

100W100Na

Amtmann er a / . (1997)

106 W32 Na 100 Nd100 K

Roberts and Tester (1997a) Elzenga and Van Volkenburgh (1994) White and Lemtiri-Chlieh, (1995)

G(I3l

Ere"

White (1993)

100 K + 4 Nd150 K 100K+4Na/250K

( I ) impalement of intact cell. (2) fitted negative limit conductance. (3) at -120mV. (4) at -200mV. ( 5 ) at -130mV. (6) at -lSOmV, (7) at -170mV, (8) at -180mV. (9) at -230mV. (10) lipid bilayer. (11) at positive V. (12) at -40mV. (13) between -30 and +30mV

_-

88

A. AMTMANN and D. SANDERS

dependent inward current in the plasma membrane of barley suspension cultured cells. This suggestion was based on the observation of not only unusually high PNa:PK of the ‘IRC’ (Table I) but also a high variation in PNa:PK. Indeed, single channel experiments reveal the existence of more than one type of inward rectifying cation channel within the same membrane (Vogelzang and Prins, 1994; Maathuis and Sanders, 1995; Amtmann et al., 1997; Wegner and De Boer, 1997a). C . OUTWARD-RECTIFYING CHANNELS (ORCS)

In direct contrast to inward-rectifying channels, outward-rectifying channels are responsible for cation efflux from the cell. Like the IRCs, however, ORCs are time-dependent and open only over a period of hundreds of milliseconds after onset of a permissive voltage pulse. Outward-rectifying cation channels are proposed to function primarily in Kf release, thus facilitating turgor loss (for example in guard cells) and stabilization of V,,,at relatively negative values in the region of EK (Maathuis ef al., 1997). One such channel - KCOl - has, to date, been identified at a molecular level in plants (Czempinski et al., 1997). The predicted membrane disposition of KCO 1 comprises four transmembrane domains and two additional domains which might contribute to ionic selectivity by forming the pore. After heterologous expression in insect cells (SB), KCOl was found to be potently activated by cytosolic free CaZ+ in the physiological range of concentrations (150-500 nM). This supports a role for channel opening in conditions commonly associated with elevated cytosolic free Ca” and membrane depolarization, such as prevail during many signalling events (Ward et a!., 1995). ORCs are considerably more variable than IRCs in their reported relative selectivities for Na+. Thus, PNa:PK ratios vary between 0.008and 1.0 in different cell types (Table I). It has been suggested that ORCs are involved in Na uptake in saline conditions (Schachtman et al., 1991). The idea is based on the assumption that in saline conditions V,,, attains values more positive than EK, in which case the channels would open, but the electrochemical potential difference for Naf would force Na+ in through the channels while K+ is exiting. One corollary of this notion might be that salt-tolerance is achieved through decreased Na+ permeability of outward-rectifying channels, and indeed Murata et al. (1994) found that the whole cell outward K+ current in salt-adapted tobacco culture cells was lower than that in non-adapted lines. However, no such difference emerged for genotypes of wheat differing in their salt-tolerance (Schachtman et al., 1991). Furthermore, both studies show that the PNa:PK of the outward rectifier is the same in the different cell lines or genotypes. D. VOLTAGE-INDEPENDENT CHANNELS (VlCS)

In addition to the inward- and outward-rectifying cation channels, increasing numbers of studies are now reporting the presence in plant plasma membranes of

MECHANISMS OF Na' UPTAKE BY PLANT CELLS

89

cation channels which are not significantly voltage-dependent. Thus, although the currents through these channels change as a function of the voltage as a driving force, voltage has no influence over channel gating. Unlike the voltage-dependent channels, the currents through these channels change instantaneously as a function of voltage when channel activity is assayed in the whole-cell configuration (i.e. the currents are not time-dependent like those through IRCs and ORCs). None of the voltage-independent channels has been identified at a molecular level. In general, these voltage-independent channels display considerably lower selectivity for K + over Na' than even outward-rectifying channels (Table I). Indeed, in some conditions, values for PNn:PK > 1 have been reported. Clearly, these rather non-selective cation channels have the potential to function as major pathways for Na' entry in saline conditions. The physiological function of VICs is not known: it might be simply to provide sufficient background conductance to allow continuous operation of the electrogenic H' pump. In the context of salinity, an important role for VICs may be to mediate voltage-independent cation uptake at increasing external osmotic pressure and thus allow quick adjustment of turgor regardless of the actual membrane potential. However, the Na ' leak provided by such channel type may be a problem for the cell in the long term. As discussed by White and Ridout (1995) these channels would have to be down-regulated if excessive Na' uptake in saline conditions is to be avoided (see below). So far non-selective VICs have been described only for glycophytic plants. VICs are quite distinct from voltage-dependent channels with respect to their inhibitor spectrum. For the three general classes of cation channel discussed so far, Table I1 summarizes the response to several inorganic ions and organic compounds. A common feature of VICs is their lack of sensitivity towards classical blockers of rectifying K' currents such as TEAC and Cst. Interestingly, the few exceptional examples of IRCs and ORCs which are not sensitive to classical blockers are those with atypically high Na+:K' selectivity. Thus, the non-selective 'spiky' inward rectifier of wheat root cells (Tyerman et a/., 1997) is not blocked by TEA' and Cs' , and the non-selective outward rectifier of barley root cells (NORC, Wegner and Raschke, 1994) lacks sensitivity towards TEA'. Blockage by TEA' and Cs' could therefore be used to pharmacologically separate K' -selective and nonselective channels. By contrast, verapamil and tetrodotoxin (TTX) which have been reported to inhibit **Na' uptake into wheat root membrane vesicles (Allen et al., 1995) failed to reduce instantaneous Na' current in wheat root protoplasts (Tyerman et al., 1997) or single channel Na+ currents in maize root protoplasts (Roberts and Tester, 1995).

E. CO-RESIDENCY OF DIFFERENT CHANNEL TYPES

In a large number of studies on plant plasma membrane cation-selective channels, the issue of Na' permeation has been investigated almost incidentally to that of K'

TABLE II Inhibitor pmjile of plant plasma membrane cation channels Channel

~

_ __

TEA+

_

cs+

Quinidine

Ba2+

Verapamil

-

IRC ORC

Yes Yes

Yes No Yes*

VIC

No

No

References

.- .

Yes Yes

No No Yes*

Yes**

Yes

Yes

No**

No*

TTX

No***

For review see White (1997) For review see White (1997); also: Maathuis and Sanders (1995)*, Schauf and Wilson (1987b)*, Tyerman et al. (1997)** White and Lemtiri-Clieh (1995), Elzenga and Van Volkenburgh (1994)*, Tyerman et al. (1997)**, Roberts and Tester (1997a)***

MECHANISMS OF Na' UPTAKE B Y PLANT CELLS

91

permeation, which usually forms the primary study subject. Furthermore, in many cases, the experimental aims have been to characterize single channel types rather than to assay the relative contributions of different channel types to the overall ion flux. Three recent studies have specifically addressed the roles of different classes of ion channel in Nat permeation in different cell types: maize root cortical cells (Roberts and Tester, 1997a); wheat root cortical cells (Tyerman et al., 1997); and suspension cell cultures derived originally from the scutelli of immature barley embryos (Amtmann et al., 1997). All three studies have reported that, to varying extents, voltage-dependent and -independent cation channels can co-reside in the same membrane (Table 111). Whether the variable frequency of co-residence is a facet of differential expression or of post-translational control is not known. However, the findings are of immense significance when viewed in the context of the widely differing PNu:PK values of IRCs, ORCs and VICs (Table I). Thus, it can be anticipated that considerable control can be exerted on the Na+ and K+ permeabilities of the plasma membrane by controlljng the relative activities of defined channel types.

V. CONTRIBUTIONS OF DIFFERENT CHANNEL-TYPES TO Na+ ENTRY IN PHYSIOLOGICAL CONDITIONS A. SEMIQUANTITATIVE DISSECTION OF FLUXES

From the data presented in the previous section, it can be concluded that several channel types are likely to contribute to the uptake of Na', even within a single cell. With respect both to the control of Na' entry in normal cells and to the identification of regulatory sites in the engineering of salinity tolerance, it is essential to establish first how much total Na+ enters the cell through the ensemble of different channel types in physiological conditions, and second what is the relative contribution of each channel type to total Na' entry. In most studies the absolute amount of current through inward rectifiers - once activated - has been reported as larger than the amount of current through the voltage-independent channels (Amtmann et al., 1997; Roberts and Tester, 1997a; Tyerman et al., 1997). However, in reaching an assessment of the respective physiological roles of these channels, one must take into account that patch clamp studies of the K*-selective rectifiers generally use unphysiologically high external K+ concentrations (between 30mM and 120mM in the studies cited above). K + concentrations at the root surface in soils are normally at least 50 times smaller than these values (Maathuis and Sanders, 1996) and apoplastic K+ concentrations in leaves, although higher, are nevertheless about five times smaller than Na+ concentrations when plants are exposed to salinity (Bernstein, 1971; Hajibagheri et af.,1987; Flowers et al., 1991; Speer and Kaiser, 1991). We have therefore employed known permeability and gating properties of the distinct classes of cation channels to visualize their respective contributions to plasma membrane Na' and K+ fluxes in various external conditions. The three

92

A. AMTMANN and D. SANDERS

TABLE 111 Cation injli4.x channels in maize, wheat and barley protoplasts

Origin of protoplasts

Whole cell currents .

~

Maize root cortex“’

Wheat root cortex‘2’

Time-dependent inward current “KIR” - in 95% of protoplasts Instantaneous current - in 50% of protoplasts Time-dependent inward current “KIR” - in 23% of protoplasts Spiky inward rectifier - in 68% of protoplasts Instantaneous current “FNa” - in 100% of protoplasts Time-dependent current “SNa” - in some protoplasts

Barley Time-dependent inward suspension current “KIR” - in ~ulture‘~’ 100% of protoplasts Time-dependent inward current - in 65% of protoplasts Instantaneous current - in 100% of protoplasts

Permeability for Na+

Single channels ~~~

~

~

~

‘‘KIR”

LOW

- 24 PS (120/100 K)

“Na-permeable channel” - 15 pS (120/100Na)

High Low

High “Noisy channel” -30 pS (10 W10 K + 100 Na)

High High

“KIR” - 12pS (100/100K)

Low

50 pS channel‘?

High

“Microchannel”? Non-selective cation channel -7 pS (100/100 K)

High

“’Roberts and Tester ( I 9951, Roberts and Tester (1997a). ‘“Findlay et (11. (1994), Tyerman et al. ( I 997). ‘”Amtmann er al. (1997)

channel types considered are the following: a hyperpolarization-activated IRC with a PNa:PK= 0.02:l; a depolarization-activated ORC with a PNa:PK = 0.l:l;and a VIC with a PNa:PK = 1:l. All channels are assumed to obey the GoldmanHodgkin-Katz current equation (see section 1V.A and legend to Fig. 2). To obtain the respective channel-mediated currents across the membrane of the whole cell, the currents predicted by the GHK equation are multiplied by the total number of channels per cell and, for the voltage-dependent channels, a Boltzman distribution of the open probability (Po:equations and parameter values are given in the legend of Fig. 2). We have taken account of the relative size of time-dependent and instantaneous currents determined in patch clamp experiments (for example in Maathuis and Sanders, 1995; Roberts and Tester, 1995; White and Lemtiri-Clieh, 1995; Amtmann et al., 1997) by assuming a relatively large number of IRCs, a

93

MECHANISMS OF Nat UPTAKE B Y PLANT CELLS

B

A

200

-

........Na'arrents

-

-200 1 I /pA

-200 I IpA

Fig. 2. Model current-voltage relations for K+ and Na+ passage through three types of cation channels in high-salt conditions. A: K'-inward rectifier (IRC) and K+-outward rectifier (ORC). B: Voltage-independent non-selective channel (VIC). EK and ENaare the equilibrium potentials for K' and Na' under the given conditions. EIRc,EoRC and EVlCare the reversal potentials of the open channel current carried by K+ and Na+ through the indicated channel type. External and cytosolic ion activities on which the example is based are indicated in the upper part of the figure. The individual I-V relations have been obtained with the Goldman-Hodgkin-Katz equation which is multiplied with the open probability, Po, and the total number of channels, N. Thus,

I(V) = P,,NP(F'/RT)V(Si- S, exp(-FV/RT))/(I

- exp(-

FVIRT))

with P , = ( I - 141 + exp(-z,(F/RT)(V - Vso))) for the IRC, f, = 1/(1 + exp(-z, (F/RT(V - Vs,,)))2for the ORC and Po = 1 for the VIC (compare White, 1997). R, T and F have their usual meanings, P is the permeability of the channel for a certain ion, S, and S, are the cytosolic and external activities, respectively, of the ion, z, is the gating charge and V,,, is the half-activation potential. The following parameters were chosen to generate the m3 s-' (for Na'), z = 2 and curves: IRC: Nf = 2 X lo-'' m3 s'-' (for K+) or 4 X Vso= -1SOmV ORC: N P = 11.2SX I O - ' * d s - ' (for K+) or 11.25X 10-'9m's-' (for Na'), z, = 1 and V , , = +35 mV; VIC: Nf = lo-'' m3 s-' (for K + and Na'). The values of the product Nf can be interpreted as 300 active IRCs with a single channel conductance ( g ) of 25 pS in l00mM K', 120 active ORCs with g = 40pS and 20 active VICs with g = 20pS. Total currents through an individual channel were obtained by adding the respective K+- and Na+-currents.

94

A. AMTMANN and D. SANDERS

moderate number of ORCs and a small number of VICs (see legend of Fig. 2). The values for relative permeabilities, single channel conductances, half-activation potentials and gating charges have been chosen as being representative for these channel types (compare Table I, Roberts and Tester, 1995 and White, 1997). To maximize the information from this exercise, we present the results as current-voltage (I-V) relationships: for those not used to thinking in these electrophysiological terms, it merely has to be recalled that the currents are directly proportional to the flux, and that the I-V relationship therefore shows how this flux varies as a function of the prevailing V,. Currents below the voltage axis are negative and inward: they represent a flow of cations into the cell. The converse holds for currents above the voltage axis. Figs 2 and 3 show examples of model currents through the three classes of channel in high-salt and low-salt environments. A high-salt environment is simulated with a K+ concentration of 4 mM and a Na+ concentration of 200 mM; low-salt conditions are represented with 1 mM Kf and 1 mM Na'. The cytosol is assumed to contain 100 mM K+ and 20 mM Na+ or 1 O O m M Kf and 1 mM Na' in high-salt or low-salt conditions, respectively. The resulting equilibrium potentials (E,",,)are EK= -81 mV and ENa= +58 mV for high-salt, and EK= - 116 mV and ENa = 0 mV for low-salt conditions. Fig. 2 depicts the Kf and Na' currents passing through each of the three channels in high-salt conditions (IRC and ORC in A, VIC in B). In a high-salt environment IRCs provide similar uptake rates for K+ and Na', despite their strong selectivity for K + over Na'. This is due both to the high Na' concentration in the medium and to the voltage range of activation being far away from ENa.thereby evoking maximum conductance. Conversely, in the same conditions Na+-uptake through ORCs is small and not discernible on Fig. 2A, even though these channels are only moderately selective for Kf over N a t . This result can be explained because the voltage range of activation is close to ENa. Importantly, Fig. 2B demonstrates that VICs mediate larger Na' influx at all physiological (negative) values of V,,, than do the voltage-dependent channels, despite the fact that the total number of active channels is taken to be only 7% and 17% of the corresponding values for IRCs and ORCs, respectively (Fig. 2, legend). The high uptake of Na' via the VICs arises because of their high permeability for Na' and the fact that they remain open over the whole voltage range. Fig. 3 shows the contribution of Kf and Na' currents to the total current in (A) high-salt and (B) low-salt conditions. The total current in high-salt conditions is the sum of the currents shown in Fig. 2. The ratio of Na+ current: K' current becomes smaller on membrane hyperpolarization due to activation of highly K+-selective IRCs (see also Fig. 4), but it is important to note that in saline conditions the absolute amount of Na+ inward current at very negative potentials is still much larger than the absolute K+ current. Fig. 3B demonstrates that Na' influx through both a small number of non-selective VICs and a large number of highly-selective IRCs becomes insignificant as the Na' and K+ concentrations in the soil drop to non-saline values. We therefore arrive at the following important conclusion: although the contribu-

95

MECHANISMS OF Na' UPTAKE BY PLANT CELLS

B

A 4 mM K'

1 rnM K'

0 total Na'current

vrn 0

-2

-400J I /PA

0

-400.I I IpA

Fig. 3. Contribution of K+-current and Na+-currents to the total current through all three channel types (IRC, ORC, VIC) in high-salt (A) and low-salt (B) conditions. External and cytosolic ion activities are given in the upper part of the figure. K+-(Na+-)currents and the total current were obtained by adding the corresponding currents through the individual channel types shown in Fig. 2. For low-salt conditions V,, was taken as - 180 mV for the IRC and 0 m V for the ORC.

tion of VICs to the whole-cell current in low-salt conditions is negligible, this class of ion channels comprises the dominant element of Na+ currents in high-salt conditions. B. RELATIVE ACTIVITY OF DIFFERENT CHANNEL TYPES DETERMINES RATE OF Na' UPTAKE

The existence of several classes of cation influx channel with different Na+:K+ discrimination profiles allows modulation of Na' uptake depending on the relative activity of the different channel types. Indeed, variable Na' currents have been described for maize, wheat and barley protoplasts and these can be correlated with the manifestation of the respective channels which, to different degrees, allow permeation of Nat (Roberts and Tester, 1995, 1997a; Tyerman ef al., 1997; Amtmann et al., 1997). An important question is whether the variation in Na+ uptake via ion channels is purely random - implying that Na+ homeostasis and salt tolerance is achieved by flexible export mechanisms - or whether Na' uptake itself is controlled via differential regulation of the ion channels involved. Although there

96

A. AMTMANN and D. SANDERS

1 : 1

-200

V/,

mV

-100

Fig. 4. Na+:K'' permeability of a membrane containing IRC and VIC (parameters as in Figs 2 and 3) as a function of voltage in high-salt and low-salt conditions (ion concentrations as in Fig. 3). The dotted line indicates similar permeability for both ions, values below this line represent higher permeability for K+ than Na+, values above the line represent higher permeability for Na+ than K*.

are indications that salt-tolerance is linked to regulation of Na' uptake rather than Na' efflux (Schubert and Lauchli, 1990), this issue remains open until all pathways involved in Na' transport have been characterized in detail. It has been shown, though, that some channels involved in Na' uptake do respond to certain putative regulators (see below). However, in most cases it is not possible to distinguish whether this regulation should be viewed in the context of salt stress, or whether the resulting change in Na' uptake is a secondary effect of channel regulation which is not primarily linked to salt stress. This distinction becomes particularly blurred when it is appreciated that the physiological functions of VICs are not fully understood. In order to determine whether regulatory features of a certain channel type are directly linked to salt stress, it will be necessary to compare salt-sensitive and salt-tolerant genotypes.

VI.

REGULATION OF MONOVALENT CATION INFLUX ACROSS THE PLASMA MEMBRANE

Table IV summarizes the current knowledge about external and cytosolic regulation of cation uptake channels in plant plasma membranes. A fair amount of data are available for IRCs, whereas regulation of VICs has been addressed in only a few studies.

MECHANISMS OF Na' UPTAKE BY PLANT CELLS

97

A. VOLTAGE

The most obvious regulatory factor of relative cation influx is the membrane voltage, V,,,. All ion currents are directly dependent on the prevailing V, because it sets the driving force for ion movement. So-called voltage-dependent ion channels such as hyperpolarization-activated IRCs are additionally voltage-dependent in their gating. The difference between lRCs and VICs in both voltage-dependence and Na':K+ permeability results in changing overall Na+:K+ permeability of the membrane with V,,, when both channel types are present. This is exemplified in Fig. 4, which shows Na+:K+ permeability of the plasma membrane calculated for a voltage range between - 120 and - 190 mV, for low-salt and high-salt conditions using the same parameters as in the model calculations described previously (see Figs 2 and 3). There is a sharp increase of relative Na':K+ permeability at voltages positive of - 160 mV (high salt) or - 140 mV (low salt) rising from about fourfold at - 180 mV to more than 20-fold at - 120 mV in high salt conditions and from negligible values at - 180 mV to about fourfold at - 120 mV in low-salt conditions. Of course, V,,, is itself regulated by the amount and the type of ion channels open, as channel currents off-set the hyperpolarizing action of the ATP-consuming H + pump and drive the prevailing V,, towards the equilibrium potential of the permeant ions. The effect of raising external Na' on V,, is variable and possibly dependent on cell type. Some studies have suggested a depolarization upon addition of external Na" (root cortical cells of sunflower, Cakirlar and Bowling, 1981; NitelEopsis, Katsuhara and Tazawa, 1990; Charu injam, Kourie and Findlay, 1990), whereas others report either no effect or a transient one (Acetabularia, Amtmann and Gradmann, 1994; root cells of maize, Cocucci et al., 1976; Cheeseman, 1982). In salt-adapted barley suspension cultured cells no statistical difference was found between the membrane potentials of protoplasts bathed in 10 mM KCI and those exposed to 1 O m M KCI + 150mM NaCl (Amtmann er al., 1997). In both conditions about 50% of protoplasts displayed membrane potentials considerably more negative than Err and EN.,. More detailed information about V,,, in various conditions is required in order to determine the resulting relative activity of different ion channel types and thus the relative amount of Nat and Kt uptake. It is clear, however, that although V,,, can regulate the Na+:K' influx ratio over a wide range (Fig. 4), it will never be able to reduce Na' uptake dramatically since the channel type that is mainly responsible for Na* uptake is voltage-independent. In order to close non-selective VICs effectively regulators other than voltage are needed. B.

EXTERNAL CALCIUM AND pH

Increasing external Ca2+ concentrations have been shown to relieve salt-stress symptoms, principally by decreasing the ratio of Na+:K+ uptake (Cramer el a/., 1987; Lauchli, 1990; Rengel, 1992). Several IRCs are blocked by high external Ca" (Table IV). However, since IRCs display the lowest PN,:P, values reported

TABLE IV Regulation of cation uptake channels in plant plasma membranes (+: activation, -: inhibition, 0 no effect) Regulator

IRC

VIC

ChanneVCell type

References

-.

~~

+

Membrane voltage (hyperpolarization)

All cell types 0'')

Schroeder er al. (1994), Hedrich and Becker (1994) Maathuis et al. (1997) (reviews) White and Lemtiri-Clieh (1995), Roberts and Tester (1997), Tyerman et al. (1997), Elzenga and van Volkenburgh (1994), Amtmann et al. (1997)

Root (rye, maize, wheat) leaf mesophyll (pea) suspension culture (barley)

-

External Ca2+

-

-

~-

Guard cells (Ecia), Root (barley, maize, wheat) Coleoptiles (maize) Root (maize, wheat) _-

External H+ ~.

-

Blatt (1992), Fairley-Grenot and Assmann (1992), Wegner et al. (1994), Roberts and Tester (1995) Tyerman ef al. (1997) Thiel et al. (1996) Roberts and Tester (1997b), Tyerman et al. (1997) . -

_______._

Guard cells (Ecia), KAT1, KSTl

Blatt ( 1992) Hedrich et al. (1995a), Wry et al. (1995), Miiller-Rober et al. (1995)

0

Root (barley, wheat) KAT 1 Guard cells (Ecia, potato,

Wegner et al. (1994), White and Lemtiri-Clieh (1995) Hoshi (1995), Marten er al. (1996) Hedrich and Dietrich (1996) (review)

._

Cytoplasmic Ca2+

-

1-

-._.

Cytoplasmic H+

______-

+

+

Coleoptiles (maize) Leaf mesophyll (pea)

Hedrich et al. (1995b) Elzenga and van Volkenburgh (1994)

Guard cells (Ecia) KATl

Blatt (1992) Hoshi (1995)

. .-

0

+

-

External Na+

0

+ Cytoplasmic Na+

0 0

Cham,mesophyll (oat), root (barley) Root (wheat), suspension culture (barley), KATl AKTl

Tester (1988); Kourie and Goldsmith (1992); Wegner and Raschke ( 1994) Tyerman et al. (1997), Amtmann et al. (1997); Bertl et ul. (1995) Bertl e l ul. (1997)

Suspension culture (barley), root (maize) Root (maize)

Amtmann et al. (1997); Roberts and Tester (1997a)

. -~ ~~~~~

ATP

-k

+ -

Protein phosphatases (dephosphorylation) ~~~

G-proteins

Roberts and Tester ( I 997a) ~

~~

~~

-

+ + -

Guard cells (Vicia) KATl, KSTl Suspension culture (barley) Suspension culture (barley) Nitellopsis obtusa

Wu and Assmann (1995); Hoshi (1995); Miiller-Rober er al. (1995); Amtmann er al. (1997)

Guard cells (Vicia) Guard cells (Vicia)

Luan et al. (1993) Thiel and Blatt (1994); Li et al. (1994)

~~

~~~

Guard cells (Vicia) Guard cells (Viciu)

Amtmann et al. ( 1 997) Katsuhara et al. (1990)

~

_

~

~~

~

~~~

.-

Kelly el al. (1995); De Boer and Wegner (1997); Wegner and De Boer (1997a) Fairley-Grenot and Assmann (1991); Armstrong and Blatt ( 1995)

“’No voltage-dependence of gating but in some cases slight rectification of open-channel currents +, activation; -. inhibition; 0, no effect.

100

A. AMTMANN and

D.SANDERS

for plant channels (Table I), this block would increase rather than decrease the ratio of Na+:K+ uptake. Furthermore, Ca2+ inhibition of IRCs is usually weak (half maximum inhibition at [Ca"] > 10 mM) and voltage-dependent (manifesting itself only at voltages more negative than - 150 mV: Wegner et al., 1994; Roberts and Tester, 1995, but compare Thiel ef al., 1996). Recent studies on rye, maize and wheat cortical protoplasts (White and Lemtiri-Clieh, 1995; Roberts and Tester, 1997a; Tyerman et al., 1997) report that external Ca2+ blocks not only the IRCs in these cells, but also the Na+-permeable VICs. In maize and wheat protoplasts Ca" inhibition of the instantaneous inward current was measured in hyperpolarizing conditions (at - 180 mV in maize and around - 120 mV in wheat) in external solutions containing high concentrations of Na' (100 mM). The two cell types deliver strikingly similar results. In both studies maximum inhibition is SO%, indicating that the Na+-permeable pathway comprises a Cat -sensitive and a Ca2+-insensitive component, and half-maximal inhibition is achieved with a Ca2+ activity of about 300p.M (350p.Min maize, 310pM in wheat). Furthermore, single channel experiments on excised patches of maize protoplasts show that the open channel current, as well as the open probability of the Na' permeable channel, decrease when the external Ca2+ concentration is raised. In contrast, noise analysis of the whole-cell Na' current in wheat suggests that here Ca2+ affects the open channel conductance only. These findings are crucial for demonstrating the relevance of VlCs to Na' uptake. In both wheat and maize the Ca"-dependence of the Na+ currents matches very well the data obtained in "Na+-influx experiments (maize: Zidan et al., 1991; wheat: Davenport et al., 1997), thus indicating that the Na+ currents observed in patch clamp experiments are indeed responsible for the bulk of Na+ uptake. A similar point has been made by Tyerman and Skerrett (1998). Although the dependence of VIC currents on the external Ca2+ concentration is one factor which determines the ratio of Na+:K+ influx, the significance of the Ca2' sensitivity of VICs with respect to physiological regulation may be limited since the apoplastic Ca" concentration will mostly passively follow the external conditions. External protons are more likely to act as a physiological regulator of ion transport than external Ca2' , since the apoplastic pH - at least in the close vicinity of the plasma membrane - is usually determined by the activity of the plasma membrane H+-ATPase, rather than the soil pH. It is likely that the outer surface of the plasma membrane of root cells is exposed to relatively acid pH even in saline soils with neutral or basic pH. It has been shown for some plants that H ' -ATPases in the tonoplast and in the plasma membrane are up-regulated in saline environments (Braun et al., 1986; Reuveni et al., 1990; Binzel, 1995; Ayah et al., 1996). The IRCs of guard cells, as well as Arabidopsis and potato IRCs KATl and KST are activated by external H+ (via positive shift of activation potential and/or increase of maximum conductance, for references see Table I) and the same effect was measured for IRC activity in barley root cells (A. Amtmann, unpublished results). Combined action of proton pumping and IRC activation could help to

MECHANISMS OF Na' UPTAKE BY PLANT CELLS

101

improve K' uptake and thus reduce Na+:KC uptake ratio in saline conditions. Detailed studies are needed to test this hypothesis. The effect of external pH on VICs has not yet been studied. C. CYTOSOLIC CALCIUM AND pH

Lynch et al. (1989) observed an increase in cytosolic Ca2' concentration ([Ca2+],,,) after exposure of maize root protoplasts to high external NaCl concentrations (>100 mM), but it was not determined whether high Ca'+ levels were sustained or only transient. A rise of [Ca2t],,, was also found in Nitellopsis after salt exposure (100mM NaCI). Here, low resting Ca" levels were restored within 60min (Okazaki ei uf., 1996). Much shorter transient increases of [Ca'+],,, (90% recovery within 1 min) have been reported for Arubidopsis when exposed to 300 mM NaCl (Knight et a/., 1997). An increase in [Ca2+1,,, reduces currents through IRCs in guard cells. On the other hand, IRCs in root tissues appear insensitive to changes in [Ca2+lCy,.Contrastingly, a voltage-independent channel in mesophyll cells of Pisurn, which is more permeable to Nat than K' , has been found to be activated by cytoplasmic Ca" (for references see Table IV). It has yet to be verified whether this is a typical feature of non-selective cation channels. If so, a transient increase of [Ca2 Icy, would promote Naf-uptake through this channel type, whereas low resting Levels of [Ca2'Icy, would reduce non-selective channel activity while maintaining or even stimulating IRC activity. Cytoplasmic pH does not affect the IRCs in guard cells (Blatt, 1992) but is probably involved in the regulation of KATl (Hoshi, 1995). As for external pH, VICs have not yet been characterized in this respect. +

D. EXTERNAL AND CYTOSOLlC Na'

External Na+ inhibits K f inward currents in Cham and blocks the K + currents through the IRC in oat mesophyll cells (Tester, 1988; Kourie and Goldsmith, 1992). In protoplasts from barley xylem parenchyma cells external Na' was found to inhibit outward currents (tail currents) through open IRCs indicating either a reduction of the open probability or tight binding of Na' in the pore (Wegner and Raschke, 1994). In most cells blockage of IRCs in saline conditions would be problematic since it would further increase the Na ' :K' uptake ratio. In wheat root protoplasts and barley suspension cultured cells Na+ had no blocking effect on time-dependent K ' -inward currents, and neither were K' currents through KATl, when expressed in yeast, affected by Na' (for references see Table IV). However, exposure of another Arcrbidopsis K t inward rectifier expressed in yeast, AKTI, displayed larger K t currents when exposed to high external Na' concentrations and these currents were maintained for several minutes after wash-out of Na+ (Bert1 et al.. 1997). This indicates an up-regulation of AKTl by external Na', probably via tight binding of Na' to a modulation site in the channel.

102

A. AMTMANN and D. SANDERS

Cytosolic Na+, which blocks ORCs in guard cells (Thiel and Blatt, 1991) does not inhibit currents through IRCs or Na+-permeable channels. However, as in most patch clamp experiments, pH- and Ca2+-buffered pipette solutions were used in these studies; therefore an indirect effect of increased cytosolic Na+ via signalling pathways involving modulation of cytosolic Ca” or pH cannot be discounted.

E. ATP

Most patch clamp experiments on whole protoplasts include millimolar concentrations of ATP in the pipette (cytosolic) solution but very few studies have investigated whether ATP is directly necessary for channel activity. Wu and Assrnann (1995) observed in single channel experiments that the open probability of IRCs increased by a factor of five when ATP was added. Whole-cell currents through IRCs were still observed if the pipette solution did not contain ATP, but ATP-scavenging agents abolished the IRC currents. The authors concluded that only very low ATP-concentrations are needed for full activation of IRCs. In barley suspension cultured cells Amtmann et nf. (1997) detected no whole-cell IRC currents when ATP was absent from the pipette solution but large IRC currents in all experiments which included 2 mM ATP in the pipette. Washout of ATP from the pipette reduced IRC currents completely but slowly. In accord with the conclusions of Wu and Assmann (1995), this indicates that the critical ATP concentration for IRC stimulation is very low. In the same experiments about 50% of the VIC current disappeared very rapidly (A. Amtmann, unpublished results), suggesting that this current component is sensitive to millimolar, rather than micromolar, ATP concentrations. In single channel experiments a 7-pS, nonselective cation channel was indeed found to be activated by ATP (Amtmann et uf., 1997). None of the studies could distinguish between a direct effect of ATP on the channels and indirect effects via phosphorylation of the channel or membranebound regulators. Although the activities of both IRCs and VICs are dependent on ATP, modest changes or local depletion of cytosolic ATP (for example as a result of increased ATP-hydrolysis by plasma membrane and vacuolar proton pumps in high-salt conditions; see above) may selectively close VICs and thus increase the K+:Naf uptake ratio. Again, much more work is needed to test this hypothesis.

F. OTHER REGULATORS

Other factors such as protein phosphatases and G-proteins have been found to be involved in the regulation of IRCs (Table IV), but their effect on VICs remains to be elucidated. Reducing agents affect ORCs in Arubidopsis mesophyll cells (Spalding el ul., 1992), but until now no cation uptake channel has been reported to have such sensitivity.

MECHANISMS OF Na' UPTAKE BY PLANT CELLS

103

VII. COMPARISON OF SALT-SENSITIVE AND SALT-TOLERANT GENOTYPES OR CELL LINES Maintenance of a low Nat:Kt ratio in the cytosol of cells is a crucial aspect of survival for a plant in a saline environment. Some studies on the involvement of ion channels in salt-tolerance have therefore addressed the question of whether a certain channel type has a lower Na':K+ permeability ratio in salt-tolerant genotypes than in salt-sensitive genotypes (e.g. wheat: Schachtman et a/., 1991; Findlay et al., 1994). No such difference was found. Similar results emerge from the comparison of salt-adapted and non-adapted cell lines of suspension cultures (Murata et al., 1994; Amtmann et al., 1997). Whole-cell ORC currents of tobacco were decreased after adaptation (Murata et d., 1994). In barley, radiometric assays of fluxes revealed distinct uptake patterns in salt-adapted and non-adapted barley cell lines (S. Laurie and R. Leigh, personal communication). However, in both cell lines there was no significant difference either in the array of channel types present or in the basic attributes of these channel types (i.e. K ':Na+ permeability, Murata et al., 1994; Amtmann et al., 1997). As discussed previously, even a very low PNd:PKratio of IRCs would not essentially reduce Na' uptake as long as active VICs are present in the same membrane. Improvement of the selectivity of a certain channel seems not to be a reasonable way to achieve salt-tolerance, either in evolution or in biological engineering. A salt-tolerant plant will need both pathways for the uptake of Na' (which is the quickest and cheapest osmoticum in saline conditions) and other ions, and, in addition, adequate means to activate or de-activate these pathways in order to respond to the specific requirements of a cell at a given moment. The ability to compromise successfully between osmotic adjustment, ion nutrition, restriction of cytosolic Na' concentration and maintenance of energy pools is probably the key to salt-tolerance. Several different channel types facilitatjng cation uptake across the plant plasma membrane represent the necessary basis for such functional flexibility and seem to be present in most cells. Nevertheless, only those plants which can efficiently control NaC passage through these channels have a chance of survival in high-salt conditions. Analysis and comparison of channel regulation in salt-sensitive and salt-tolerant plants species must be a key issue of future studies on salt tolerance.

VIII. FUTURE WORK An integrative approach will be needed to further study Na' uptake across plant plasma membranes. So far, many of the transporters involved in Na'-uptake have been identified. Their voltage-dependence has been studied and their Na ':Kt permeability has been determined, albeit in most cases in relatively nonphysiological conditions. The following areas will need preferential attention in future experiments. The characterization of Na' uptake channels has to be extended to halophytic plants. Currents through all cation uptake channels have to be

104

A. AMTMANN and D. SANDERS

measured in physiological conditions (K', Na+, Ca2+ and pH). Interactive behaviour of relevant permeant ions has to be studied in order to design appropriate models for the calculation of the contribution of Na+ to the whole-cell current. Short-term and long-term effects of salinity on the membrane potential and putative regulators have to be determined. Special emphasis has to be put on the study of the regulation of non-selective cation channels. Once it has been revealed which are the determining factors for Na+-uptake across the plasma membrane, comparison between different cell types within the plant and between salt-sensitive and salt-tolerant species with respect to these factors should provide new insights into the complex field of salt-tolerance.

ACKNOWLEDGEMENTS We would like to thank Dr Frans Maathuis (University of York, UK) for valuable discussion and Dr Steve Qerman (University of South Australia, Adelaide, Australia) for providing us with manuscripts prior to publication. Experimental work in our laboratory was funded by the BBSRC and the EU. AA is supported by the EU.

REFERENCES Allen, G. J. and Sanders, D. (1997). Vacuolar ion channels of higher plants. Advances in Botanical Research 25, 217-252. Allen, G. J., Wyn Jones, R. G. and Leigh, R. A. (1995). Sodium transport measured in plasma membrane vesicles isolated from wheat genotypes with differing K+/Na+ discrimination traits. Plant, Cell and Environment 18, 105-1 15. Allen, G. J., Sanders, D. and Gradmann. D. (1998). Calcium-potassium selectivity: kinetic analysis of current-voltage relationships of the open slowly activating channel in the vacuolar membrane of Vicia faha guard cells. Plantu 204, 528-541. Arntmann, A. and Gradmann, D. (1994). Na+ transport in Acetabularia bypasses conductance of plasmalemma. Journal of Membrane Biology 139, 117-125. Amtmann, A., Laurie, S., Leigh, R. and Sanders, D. (1997). Multiple inward channels provide flexibility in Na+/K+ discrimination at the plasma membrane of barley suspension culture cells. Journal of Experimenral Botany 48, 48 1-497. Anderson, J. A., Huprikar, S. S., Kochian, L. V., Lucas, W. J. and Gaber, R. F. (1992). Functional expression of a probable Arabidopsis rhuliana potassium channel in Succhoromyces cerevisioe. Proceedings of the National Aradeniy of Sciences of the United States r f America 89, 313&3140. Armstrong, F. and Blatt, M. R. ( 1 995). Evidence for K+ channel control in Vicia guard cells coupled by G-proteins to a 7TMS receptor mimetic. The Plant Journal 8, 187-198. Ayah, F., O'Leary, J. W. and Schumaker, S. (1996). Increased vacuolar and plasma membrane H+-ATPase activities in Salicornia bigelovii Torr. in response to NaCI. Journal of Experimental Botany 47, 25-32. Azimov, R. R., Geletyuk, V. I. and Berestovskii, G. N. (1984). Single potential-dependent K+-channel of the cells of the alga Nitellopsis obtusa. Biojzika 32, 79-84. Barkla, B. J., Zingarelli, L., Blumwald, E. and Smith, J. A. C. (1995). Tonoplast Na+/H+

MECHANISMS OF Na' UPTAKE BY PLANT CELLS

I05

antiport activity and its energization by the vacuolar H+-ATPase in the halophytic plant Mesembtyanthemum crystallinum L. Plant Physiology 109, 549-556. Becker, D., Dreyer, I., Hoth, S., Reid, J. D., Busch, H., Lehnen, M., Palme, K. and Hednch, R. (1996). Changes in voltage activation, Cs+ sensitivity, and ion permeability in H5 mutants of the plant K' channel KATI. Proceedings of the National Academy o j Sciences of the United States of America 93, 812343128, Bernstein. L. (1971). Method for determining solutes in the cell wall of leaves. Plant Physiology 47, 361-365. Bertl, A., Anderson, J. A., Slayman, C. L. and Caber, R. E (1995). Use of Saccharomyces cerevisiae for patch-clamp analysis of heterologous membrane proteins: characterization of Katl, an inward-rectifying K* channel from Arabidopsis thaliana, and comparison with endogenous yeast channels and carriers. Proceedings of the National Academy of Sciences of the United States of America 92, 2701-2705. Bertl, A., Reid, J. D., Sentenac, H. and Slayman, C. L. (1997). Functional comparison of plant inward-rectifier channels expressed in yeast. Journal of Experimental Botany 48, 4 0 5 4 13. Binzel, M. ( 1995). NaCI-induced accumulation of tonoplast and plasma membrane H'-ATPase message in tomato. Physiologia Plantarum 94, 722-728. Binzel, M.. Hess, F. D., Bressan, R. A. and Hasegawa, P. M. (1988). Intracellular compartmentation of ion in salt adapted tobacco cells. Plant Physiology 86, 607614. Blatt, M. R. (1992). K + channels of stomata1 guard cells. Characterisation of the inward rectifier and its control by pH. Journal of General Physiology 99, 615-644. Blatt, M. R. and Gradmann, D. (1997). K+-sensitive gating of the K' outward rectifier in Vicia guard cells. Journal of Membrane Biology 158, 241-256. Blumwald, E. and Gelli, A. (1997). Secondary inorganic ion transport at the tonoplast. Advances in Botanical Research 25, 401417. Braun, Y., Hassidim, M., Lerner, H. R. and Reinhold, L. (1986). Studies on H' translocating ATPases in plants of varying resistance to salinity I. Salinity during growth modulates the proton pump in the halophyte Atriplex nummularia. Plant PhysioZoRy 81, 1050-1056. Braun, Y., Hassidim, M., Lerner, H. R. and Reinhold, L. (1988). Evidence for a Na'lH' antiporter in membrane vesicles isolated from the roots of the halophyte Atriplex nummularia. Plant Physiology 87, 104108. Bush, D. R. (1993). Proton-coupled sugar and amino-acid transporters in plants. Annual Review of Plant Physiology and Plant Molecular Biology 44,5 13-542. Bush, D. S., Hednch, R., Schroeder, J. I. and Jones, R. L. (1988). Channel-mediated K + flux in barley aleurone protoplasts. Plantu 176, 368-377. Cakirlar, H. and Bowling, D. J. F. (1981). The effect of salinity on the membrane potential of sunflower roots. Journal of Experimental Botany 32, 479-485. Cerana, R. and Colombo, R. (1992). K' and CI conductance of Arubidopsis thaliuna plasma membrane at depolarized voltages. Botanica Actu 105, 273-277. Cheeseman, J. M. (1982). Pump-leak sodium fluxes in low-salt corn roots. Journal of Membrane Biology 70. 157-1 67. Cheeseman, J. M. (1988). Mechanisms of salinity tolerance in plants. Plant Physiology 87, 547-550. Cocucci, M., Marre, E., Ballarin-Denti, A. and Scacchi, A. (1976). Characteristics of fusicoccin-induced changes of trans-membrane potential and ion uptake in maize root segments. Plant Science Letters 6, 143-156. Colombo, R. and Cerana, R. (1991). Inward rectifying K' channels in the plasma membrane of Arabidopsis thalianu. Plant Physio1og.y 97, 1130-1 135. Colombo, R., Bonetti, A. and Lado, P. (1979). Promoting effect of fusicoccin on Na+ efflux

106

A. AMTMANN and

D.SANDERS

in barley roots: evidence for Na+-H+ antiport. Plant Cell and Environment 2, 281-285. Cramer, G. R., Lynch, J., Lauchli, A. and Epstein, E. (1987). Influx of Na+, K+, and Ca2+ into roots of salt-stressed cotton seedlings. Plant Physiology 83. 5 10-5 16. Czempinski, K., Zimmermann, S., Ehrhardt, T. and Miiller-Rober, B. (1997). New structure and function in plant K+ channels: KCOl, an outward rectifier with a steep Ca" dependency. EMBO Journal 16,2565-2575. Daram, P., Urbach, S., Gaymard, F., Sentenac, H. and ChCrel, I. (1997). Tetramerization of the AKTl plant potassium channel involves its C-terminal cytoplasmic domain. EMBO Journal 16, 3455-3463. Davenport, R. J., Reid, R. J. and Smith, F. A. (1997). Sodium-calcium interactions in two wheat species differing in salinity tolerance. Physiologia Plantarum 99,323-327. De Boer, A. H.and Wegner, L. H. (1997). Regulatory mechanisms of ion channels in xylem parenchyma cells. Journal of Experimental Botany 48,441-449. Ding, L. and Zhu, J.-K. (1997). Reduced Na' uptake in the NaC1-hypersensitivesosl mutant of Arabidopsis thaliana. Plant Physiology 113,795-799. Dodd, W. A., Pitman, M. G. and West, K. R. (1966). Sodium and potassium transport in the marine alga Chaetomorpha darwinii. Australian Journal of Biological Sciences 19, 341-354. Elzenga, J. T. M. and Van Volkenburgh, E. (1994). Characterization of ion channels in the plasma membrane of epidermal cells of expanding pea (Pisum sativum arg) leaves. Journal of Membrane Biology 137. 227- 235. Epstein, E. (1961). The essential role of calcium in selective cation transport by plant cells. Plant Physiology 36,437-444. Epstein, E.,Rains, D. W. and Elzam, 0. E. (1963). Resolution of dual mechanisms of potassium absorption by barley roots. Proceedings of the National Academy of Sciences of the United States of America 49,684-692. Fairley-Grenot, K. and Assmann, S. M. (1991). Evidence for G-protein regulation of inward K+ channel current in guard cells of fava bean. Plant Cell 3, 1037-1044. Fairley-Grenot, K. A. and Assmann, S. M. (1992). Permeation of Ca2+through K+ channels in the plasma membrane of vicia faba guard cells. Journal of Membrane Biology 128, 103-113. Felle, H. H. (1994). The H+/CI- symporter in root-hair cells of Sinapis alba. Plant Physiology 106, 1131-1136. Findlay, G. P., Tyerman, S. D., Garrill, A. and Skerrett, M. (1994). Pump and K+ inward rectifiers in the plasmalemma of wheat root protoplasts. Journal of Membrane Biology 139, 103-116. Flowers, T. J. and Yeo, A. R. (1995). Breeding for salinity resistance in crop plants: Where next? Australian Journal of Plant Physiology 22, 875-884. Flowers, T. J., Troke, P. F. and Yeo, A. R. (1977). The mechanisms of salt tolerance in halophytes. Annual Review of Plant Physiology and Plant Molecular Biology 28, 89-121. Flowers, T. J., Hajibagheri, M. A. and Yeo, A. R. (1991). Ion accumulation in the cell walls of rice plants growing under saline conditions: evidence for the Oertli hypothesis. Plant Cell and Environment 14, 3 19-325. Garbarino, J. and DuPont, F. M. (1988). NaCl induces a Na+/H+ antiport in tonoplast vesicles from barley roots. Plant Physiology 86,231-236. Garbarino, J. and DuPont, F. M. (1989). Rapid induction of Na+/H+ exchange activity in barley root tonoplasts. Plant Physiology 89, 1 4 . Ganill, A., Tyerman, S. D. and Findlay, G. P. (1994). Ion channels in the plasma membrane of protoplasts from the halophytic angiosperm Zostera muelleri. Journal of Membrane Biology 142,381-393.

MECHANISMS OF Na' UPTAKE BY PLANT CELLS

I07

Gassman, W. and Schroeder, J. I. (1994). Inward- rectifying K' channels in root hairs of wheat. Plant Physiology 105, 1399-1408. Gassmann, W., Rubio, F. and Schroeder, J. I. (1996). Alkali cation selectivity of the wheat root high-affinity potassium transporter HKTI . The Plant Journal 10, 869-882. Gorham, J. (1992). Salt tolerance of plants. Science Progress 76, 273-285. Grabov, A. M. (1990). Voltage-dependent potassium channels in the root hair plasmalemma. Soviet Plant Physiology 37, 242-250. Gradmann, D. (1 996). Selectivity of ion channels: competitive analysis versus independent electrodiffusion. Journal of Experimental Botany 47, 1733-1 736. Gradmann, D., Johannes, E. and Hansen, U. P. (1997). Kinetic analysis of Ca'+/K+ selectivity of an ion channel by single-binding-site models. Journal of Membrane Biology 159, 169-178. Greenway, H. and Munns, R. (1980). Mechanisms of salt tolerance in nonhalophytes. Annual Review of Plant Physiology and Plant Molecular Biology 31, 149-190. Hajibagheri, M. A,, Harvey, D. M. R. and Flowers, T. J. (1987). Quantitative ion distribution within root cells of salt-sensitive and salt-tolerant maize varieties. The New Phytolog ist 105, 3 67-3 79. Hajibagheri, M. A. and Flowers, T. J. (1993). Use of freeze-substitution and molecular distillation drying in the preparation of Dunaliella pawa for ion localization studies by X-ray microanalysis. Microscopy Research and Technique 24, 395-399. Hamill, 0. P., Marty, A., Neher, E., Sakman, B. and Sigworth, F. J. (1981). lmproved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pjiigers Archiv 391, 85-1 00. Haro, R., Garciadeblas, B. and Rodriguez-Navarro, A. (1991). A novel P-type ATPase from yeast involved in sodium transport. FEBS Letters 291, 189-191. Hedrich, R. and Becker, D. (1994). Green circuits - The potential of plant specific ion channels. Plant Molecular Biology 26, 1637-1650. Hedrich, R. and Dietrich, P. (1996). Plant Kf channels: Similarity and diversity. Botanica Acta 109, 94-101. Hedrich, R. and Schroeder, J. I. (1989). The physiology of ion channels and electrogenic pumps in higher plants. Annual Review c$ Plant Physiology und Plant Molecular Biology 40, 539-569. Hedrich, R., Moran, O., Conti, F., Busch, H., Becker, D., Gambale, F., Dreyer, I., Kiich, A,, Neuwinger, K. and Palme, K. (1995a). Inward rectifier potassium channels in plants differ from their animal counterparts in response to voltage and channel modulators. Europeun Biophysical Journal 23, 107-1 15. Hedrich, R., Bregante, M., Dreyer, I. and Gambale, F. (1995b). The voltage-dependent potassium-uptake channel of corn coleoptiles has permeation properties different from other K' channels. Plunta 197, 193-199. Hille, B. (1992). "Ionic Channels of Excitable Membranes", 2nd edn. Sinauer, Sunderland, MA. Hoshi, T. (1995). Regulation of voltage-dependence of the KATl channel by intracellular factors. Journal of Generul Physiolqy 105, 309-328. Jacoby, B. and Teomy, S. (1988). Assessment of N a ' W antiport in ATP-depleted red beet slices and barley roots. Plant Science 55, 103-1 06. Jia, Z.-P., McCullough, N., Martel, R., Hemmingsen, S. and Young, P. G. (1992). Gene amplification at a locus encoding a putative N a ' W antiporter confers sodium and lithium tolerance in fission yeast. EMBO Journal 11, 1631-1640. Johannes, E. and Sanders, D. (1995). Lumenal calcium modulates unitary conductance and gating of a plant vacuolar calcium release channel. Journcil of Membrane Biokigy 146, 2 11-224. Katsuhara, M. and Tazawa, M. (1990). Mechanism of calcium-dependent salt tolerance in

108

A. AMTMANN and D. SANDERS

cells of Nitellopsis obtusa: role of intracellular adenine nucleotides. Plant, Cell and Environment 13, 179-184. Katsuhara, M., Mimura, T. and Tazawa, M. (1990). ATP-regulated ion channels in the plasma membrane of Characeae alga, Nitellopsis ohtusa. Plant Physiology 93, 343-346. Kelly, W. B., Esser, J. E. and Schroeder, J. I. (1995). Effects of cytosolic calcium and limited, possible dual, effects of G-protein modulators on guard-cell inward potassium channels. Plant Journal 8, 479489. Knight, H., Trewavas, A. J. and Knight, M. R. (1997). Calcium signalling in Arabidopsis thaliana responding to drought and salinity. The Plant Journal 12, 1067-1078. Kourie, J. I. and Findlay, G. P. (1990). Ionic currents across the plasmalemma of Chara infircta cells 11. Effects of external Na+, Ca” and CI- on K+ and C1- currents. Journal of Experimental Botany 41, 151-163. Kourie, J. I. and Goldsmith, M. H. M. (1992). Kf channels are responsible for an inward rectifying current in the plasma membrane of mesophyll protoplasts of Avena safiva. PIant PhysioZogy 98, 1087-1097. Krysan, P. J., Young, J. C., Tax, F. and Sussman, M. R. (1996). Identification of transferred DNA insertions within Arabidopsis genes involved in signal-transduction and ion-transport. Proceedings of the National Academy of Sciences of the United States of America 93, 8 145-8 150. Lagarde, D., Basset, M., Lepetit, M., Conejero, G., Gaymard, F., Astruc, S. and Grignon, C. (1996). Tissue-specific expression of Arabidopsis AKTl gene is consistent with a role in K+ nutrition. The Plant Journal 9, 195-203. Lass, B. and Ullrich-Eberius, C. I. (1984). Evidence for proton sulfate cotransport and its kinetics in Lemna gibba G. Planta 161, 53-60. Lauchli, A. (1990). Calcium, salinity and the plasma membrane. In “Calcium in Plant Growth and Development” (R. T. Leonard and P. K. Hepler, eds) pp. 2 6 3 5 . The American Society of Plant Physiologists, Rockville. Lauchli, A., Colmer, T. D., Fan, T. W.-M. and Higashi, R. M. (1994) Solute regulation by calcium in salt-stressed plants. In “Biochemical and Cellular Mechanisms of Stress Tolerance in Plants” (J. H. Cherry, ed.) pp. 443-461. Springer Verlag, Berlin. Lazof, D. and Cheeseman, J. M. (1986). Sodium transport and compartmentation in Spergularia marina. Plant Physiology 81, 742-747. Li, W., Luan, S., Schreiber, S. L. and Assmann, S. M. (1994). Evidence for protein phosphatase 1 and 2A regulation of K+ channels in two types of leaf cells. Plant Physiology 106, 963-970. Luan, S., Li, W. W., Rusnak, F., Assmann, S . M. and Schreiber, S. L. (1993). Immunosuppressants implicate protein phosphatase regulation of K+ channels in guard cells. Proceedings of the National Academy of Sciences of the United States of America 90, 2202-2206. Liittge, U. and Osmond, C. B. (1970). Ion absorption in Atriplex leaf tissue. 111. Site of metabolic control of light dependent chloride secretion to epidermal bladders. Australian Journal of Biological Sciences 23, 17- 25. Lynch, J., Polito, V. S. and LPuchli, A. (1989). Salinity stress increases cytoplasmic Ca activity in maize root protoplasts. PZant Physiology 90, 1271-1274. Maathuis, F. J. M. and Sanders, D. (1994). Mechanism of high-affinity potassium uptake in roots of Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 91, 9212-9276. Maathuis, F, J. M. and Sanders, D. (1995). Contrasting roles in ion transport of two K+-channel types in root cells of Arabidopsis thaliana. Planta 197, 456-464. Maathuis, F. J. M. and Sanders, D. (1996). Mechanisms for potassium absorption by higher plant roots. Physiologia Plantarum 96, 158-168.

MECHANISMS OF Na’ UPTAKE BY PLANT CELLS

109

Maathuis, F. J. M., Verlin, D., Smith, F. A., Sanders, D., FernBndez, J. A. and Walker, N. A. (1 996). The physiological relevance of Na+-coupled K+-transport. Planr Physiology 112, 1609-1616. Maathuis, F. J. M., Ichida, A. M., Sanders, D. and Schroeder, J. I. (1997). Roles of higher plant K+ channels. Plant Physiology 114, 1141-1149. Marschner. (1995). “Mineral Nutrition of Higher Plants”, 2nd edn. Academic Press, London. Marten, I., Gaymard, F., Lemaillet, G., Thibaud, J.-B., Sentenac, H. and Hedrich, R. (1996). Functional expression of the plant K+ channel KATl in insect cells. FEBS Letters 380, 229-232. Mennen, H., Jacoby, B. and Marschner, H. (1990). Is sodium proton antiport ubiquitous in plant cells? Journal of Plant Physiology 137, 180-1 83. Miller. (1986). “Ion Channel Reconstitution”. Plenum Press, New York. Moran, N., Fox, D. and Satter, R. L. (1990). Interaction of the depolarization-activated K’channel of Samunea saman with inorganic ions: a patch-clamp study. Plant Physiology 94, 424-43 1. Miiller-Rober, B., Ellenberg, J., Provart, N., Willmitzer, L., Busch, H., Becker, D., Dietrich, P., Hoth, S . and Hedrich, R. (1995). Cloning and electrophysiological analysis of KSTI, an inward rectifying K’ channel expressed in potato guard cells. EMBO Journal 14, 2409-24 16. Mummert, H. and Gradmann, D. (1991). Ion fluxes in Acetabularia: vesicular shuttle. Journal of Membrane Biology 124, 255-263. Munns, R. (1985). Na+, K’ and C1- xylem sap flowing to shoots of NaC1-treated barley. Journal of Experimental Botany 36, 1032-1042. Munns, R. (1993). Physiological processes limiting plant growth in saline soils: some dogmas and hypotheses. Plant, Cell and Environment 16, 15-24. Munns, R., Greenway, H. and Kirst, G. 0. (1983). Halotolerant eukaryotes. I n “Physiological Plant Ecology 111. Responses to the Chemical and Biological Environment” (0.L. Lange, P. S. Nobel, C. B. Osmond and H. Zeigler, eds) pp. 59-135. Springer, Berlin. Murata, Y., Obi, I., Yoshihashi, M., Noguchi, M. and Kakutani, T. (1994). Reduced permeability to K+ and Na+ ions of K+ channels in the plasma membrane of tobacco cells in suspension after adaptation to 50 mM NACI. Plant and Cell Physiology 35. 87-92. Nakamura, R. L., McKendree, W. L., Hirsch, R. E., Sedbrook, J. C., Gaber, R. F. and Sussman, M. R. (1995). Expression of an Arahidopsis potassium channel gene in guard cells. Plant Physiology 109, 37 1-374. Niu, X., Bressan, R. A., Hasegawa, P. M. and Pardo, J. M. (1995). Ion homeostasis in NaCl stress environments. Plant Physiology 109, 735-742. Obermeyer, G. and Blatt, M. R. (1995). Electrical properties of intact pollen grains of Ltliurn longiflorum: characteristics of the non-germinating pollen grain. Journal of Experimental Botany 46, 803-8 13. Okazaki, Y., Kikuyama, M., Hiramoto, Y. and Iwasaki, N. (1996). Short-term regulation of cytosolic Ca”, cytosolic pH and vacuolar pH under NaCI stress in the charophyte alga Nitellopsis obtusa. Plant, Cell and Environment 19, 569-576. Pick, U. (1992). ATPases and ion transport in Dunaliella. In “Dunaliella: Physiology, Biochemistry and Biotechnology” (M. Avron and A. Ben Amotz, eds) pp. 63-97. CRC Press, Boca Raton, FL. Pollard, A. and Wyn Jones, R. G. (1979). Enzyme activities in concentrated solutions of glycinebetdine and other solutes. Planta 144, 291-298. Rains, D. W. (1972). Salt transport by plants in relation to salinity. Annual Review ofPlant Physiology and Plant Molecular Biology 23, 367-388.

110

A. AMTMANN and

D.SANDERS

Rengel, Z. (1992). The role of calcium in salt toxicity. Plant, Cell and Environment 15, 625-632. Reuveni, M.,Bennett, A. B., Bressan, R. A. and Hasegawa, P. M. (1990). Enhanced H* transport capacity and ATP hydrolysis activity of the tonoplast H+-ATPase after NaCl adaptation. Plant Physiology 94, 524-530. Roberts, S. K. and Tester, M. A. (1995). Inward and outward K+-selective currents in the plasma membrane of protoplasts from maize root cortex and stele. The Plant Journal 8, 811-825. Roberts, S . K. and Tester, M.(1997a). A patch clamp study of Na+ transport in maize roots. Journal of Experimental Botany 48,43 1 4 0 . Roberts, S. K. and Tester, M. A. (1997b). Permeation of Ca2' and monovalent cations through an outwardly rectifying channel in maize root stelar cells. Journal of Experimental Botany 48, 839-846. Rubio, F., Gassman, W. and Schroeder, J. I. (1995). Sodium-driven potassium uptake by the plant potassium transporter HKT I and mutations conferring salt tolerance. Science 270, 1660-1663. Rubio, F., Gassmann, W. and Schroeder, J. I. (1996). High-affinity potassium uptake in plants - response. Science 273, 978-979. Schachtman, D. P., Tyerman, S . D. and Terry, B. R. (1991). The K+/Na+ selectivity of a cation channel in the plasma membrane of mot cells does not differ in salt-tolerant and salt-sensitive wheat species. Plant Physiology 97, 598-605. Schachtman, D. P., Schroeder, J. I., Lucas, W. J., Anderson, J. A. and Caber, R. F. (1992). Expression of an inward-rectifying potassium channel by the Arabidopsis KATI cDNA. Science 258, 1654-1658. Schachtman, D. P., Kumar, R., Schroeder, J. I. and Marsh, E. L. (1997). Molecular and functional characterization of a novel low-affinity cation transporter (LCTI) in higher plants. Proceedings of the National Academy of Sciences of the United States of America 94, 11079-11084. Schauf, C. L. and Wilson, K. J. (1987a). Effects of absicic acid on K+ channels in Viciafaba guard cell protoplasts. Biochemical and Biophysical Research Communications 145, 284-290. Schauf, C. L. and Wilson, K. J. (1987b). Properties of single K+ and C1- channels in Asclepias tuberosa protoplasts. Plant Physiology 85, 413418. Schroeder, J. I., Hedrich, R. and Fernandez, J. M. (1984). Potassium-selective single channels in guard cell protoplasts of Viciafaba. Nature 312, 361-362. Schroeder, J. I., Raschke, K. and Neher, E. (1987). Voltage-dependence of K+ channels in guard cell protoplasts. Proceedings of the National Academy of Sciences of the United Stares of America 84, 410841 12. Schroeder, J. I., Ward, J. M. and Gassman, W. (1994). Perspectives on the physiology and structure of inward-rectifying K+ channels in higher plants: biophysical implications for K+ uptake. Annual Reviews of Biophysical and Biomolecular Structure 23, 441471. Schubert, S. and Uuchli, A. (1990). Sodium exclusion mechanism at the root surface of 2 maize cultivars. Plant and Soil 123, 205-209. Sentenac, H., Bonneaud, N., Minet, M., Lacroute, F., Salmon, J.-M., Gaymard, F, and Grignon, C. (1992). Cloning and expression in yeast of a plant potassium ion transport system. Science 256, 663-665. Serrano, R. (1996). Salt tolerance in plants and microorganisms: toxicity targets and defense responses. International Review of Cytology 165, 1-52. Smith, F. A. and Walker, N. A. (1989). Transport of potassium in Chara australis: I. A symport with sodium. Journal of Membrane Biology 108, 125-137. Spalding, E. P., Slayman, C. L., Goldsmith, M. H. M., Gradmann, D. and Bertl, A. (1992).

MECHANISMS OF Na+ UPTAKE B Y PLANT CELLS

111

Ion channels in Arabidopsis plasma membrane: transport characteristics and involvement in light-induced voltage changes. Plant Physiology 99, 96-102. Speer, M. and Kaiser, W. M. (1991). Ion relations of symplastic and apoplastic space in leaves from Spinacia oleracea L. and Pisum sativum L. under salinity. Plant Physiology 97, 990-997. Stein, W. D. (1990). “Channels, Carriers and Pumps”. Academic Press, Inc., San Diego, California. Stoeckel, H. and Takeda, K. ( 1989). Calcium-activated, voltage-dependent, non-selective cation currents in endosperm plasma membrane from higher plants. Proceedings of the Royal Society of London B 237, 2 13-23 1. Szabolcs, I. (1987). The global problems of salt-affected soils. Acfa Agronomica Hungarica 36, 159-172. Terry, B. R., Tyerman, S. D. and Findlay, G. P. (1991). Ion channels in the plasma membrane of Amaranthus protoplasts: one cation and one anion channel dominate the conductance. Journal of Membrane Biology 121, 223-236. Tester, M. A. (1988). Blockade of potassium channels in the plasmalemma of Cham comllina by tetraethylammonium, Ba2+, Na2+ and Cs+. Journal of Membrane Biology 105, 77-85. Thiel, G. and Blatt, M. R. (1991). The mechanism of ion permeation through K+ channels of stomata1 guard cells: voltage-dependent block by Na’. Journal of Plant Physiology 138, 326-334. Thiel, G. and Blatt, M.R. (1994). Phosphatase antagonist okadaic acid inhibits steady-state K+ currents in guard cells of Vicia faba. Plant Journal 5, 727-733. Thiel, G., Briidern, A. and Gradmann, D. (1996). Small inward rectifying K’ channels in coleoptiles: Inhibition by external Ca2+ and function in cell elongation. Journal .f Membrane Biology 149, 9-20. Tyerman, S. D., Skerrett, M., Garrill, A,, Findlay, G. P. and Leigh, R. A. (1997). Pathways for the permeation of Na+ and CI- into protoplasts derived from the cortex of wheat roots. Journal of Experimental Botany 48, 459-480. Tyerman, S. D. and Skerrett, I. M. (1998). Root ion channels and salinity. Scientiae Horticulturae (in press). Ullrich-Eberius, C. I., Novacky, A. and Vanbel, A. J. E. (1984). Phosphate-uptake in Lemna gibba G - energetics and kinetics. Planta 161, 46-52. Uozumi, N., Gassmann, W., Cao, Y. and Schroeder, J. I. (1995). Identification of strong modification in cation selectivity in an Arubidopsis inward rectifying potassium channel by mutant selection in yeast. Journal of Biological Chemisrry 270, 24276-2428 1. Van Duijn, B., Ypey, D. L. and Libbenga, K. R. (1993). Whole-cell K’ currents across the plasma membrane of tobacco protoplasts from cell-suspension cultures. Plant Physiology 101, 81-88. Very, A.-A., Gaymard, F., Bosseux, C., Sentenac, H. and Thibaud, J.-B. (1995). Expression of a cloned plant K+ channel in Xenopus oocytes: analysis of macroscopic currents. The Plant Journal 7 , 321-332. Vogelzang, S. A. and Prins, H. B. A. (1994). Patch clamp analysis of the dominant plasma membrane K+ channel in root cell protoplasts of Plantago media L. Its significance for P and K state. Journal of Membrane Biology 141, 113-122. Wada, M., Satoh, S., Kasamo, K. and Fujii, T. (1989). Presence of a Na+-activated ATPase in the plasma membrane of the marine raphidophycean Heterosigma akashiwo. Plant and Cell Physiology 30, 923-928. Walker, N. A. and Sanders, D. (1991). Sodium-coupled solute transport in charophyte algae: a general mechanism for transport energization in plant cells? Planta 185, 443-445. Walker, N. A., Reid, R. J. and Smith, F. A. (1993). The uptake and metabolism of urea by

112

A. AMTMANN and D. SANDERS

Chara ausrralis: IV. Symport with sodium - a slip model for the high and low affinity systems. Journal of Membrane Biology 136, 263-271. Walker, N. A., Sanders, D. and Maathuis, F. J. M. (1996). High-affinity potassium uptake in plants. Science 273, 977-978. Ward, J. M., Pei, Z.-M. and Schroeder, J. I. (1995). Roles of ion channels in initiation of signal transduction in higher plants. Plant Cell 7, 833-844. Wegner, L. H. and De Boer, A. H. (1997a). Two inward K + channels in the xylem parenchyma cells of barley roots are regulated by G-protein modulators through a membrane-delimited pathway. P lunru 203, 506-5 16. Wegner, L. H. and De Boer, A. H. (1997b). Properties of two outward-rectifying channels in root xylem parenchyma cells suggest a role in K + homeostasis and long-distance signaling. Plant Physiology 115, 1707-1719. Wegner, L. H. and Raschke, K. (1994). Ion channels in the xylem parenchyma of barley roots. A procedure to isolate protoplasts from this tissue and a patch-clamp exploration of salt passageways into xylem vessels. Plant Physiology 105, 799-8 13. Wegner, L. H., De Boer, A. H. and Raschke, K. (1994). Properties of the K+ inward rectifier in the plasma membrane of xylem parenchyma cells from barley roots: Effects of TEA’, Ca”, Ba2+ and La”. Journal of Membrane Biology 142, 363-379. White, P. J. (1993). Characterization of a high-conductance, voltage-dependent cation channel from the plasma membrane of rye roots in planar lipid bilayers. Planta 191,

541-55 1. White, P. J. (1997). Cation channels in the plasma membrane of rye roots. Journal of Experimental Botany 48, 499-5 14. White, P. J. and Lemtiri-Clieh, F. (1995). Potassium currents across the plasma membrane of protoplasts derived from rye roots: a patch clamp study. Journal of Experimental Botany 46, 497-5 1 I . White, P. J. and Ridout, M. (1995). The K+ channel in the plasma membrane of rye roots has a multiple ion residency pore. Journal of Membrane Biology 143, 37-49. White, P. J. and Tester, M. A. (1992). Potassium channels from the plasma membrane of rye roots characterized following incorporation into planar lipid bilayers. Phnta 186, 188-202. Wu, W.-H. and Assmann, S. M. (1995). Is ATP required for K+ channel activation in Vicia guard cells? Plant Physiology 107, 101-109. Wu, S.-J., Ding, L. and Zhu, J.-K. (1996) SOSI, a genetic locus essential for salt-tolerance and potassium acquisition. Plant Cell 8, 617-627. Wyn Jones, R. G. and Pollard, A. (1983). Proteins, enzymes and inorganic ions. In “Encyclopedia of Plant Physiology” (A. Lauchli and A. Pirson, eds) pp. 528-562. Springer, Berlin. Yeo, A. (1988) Molecular biology of salt tolerance in the context of whole-plant physiology. Journal of Experimental Botany 49, 9 15-929. Zidan, I., Jacoby, B., Ravina, I. and Neumann, P. M. (1991). Sodium does not compete with calcium in saturating plasma membrane sites regulating **Nainflux in salinized maize roots. Plant Physiology 96, 331-334.

The NaCl Induced Inhibition of Shoot Growth: The Case for Disturbed Nutrition with Special Consideration of Calcium

DENNIS B. LAZOF' and NIRIT BERNSTEIN'

'Department of Chemistry, CB 3290, University of North Carolina, Chapel Hill,North Carolina, USA 21nstitute of Soil and Water; The Volcani Center, PO Box 6, Bet Dagan 50250, Israel

I.

Introduction to the Inhibition of Shoot Growth by Salinity ........................... A. Growth Inhibitions: General Considerations .. ............................. B. NaCI-induced Inhibition of Shoot Growth: Ge ypotheses .. C. A Nutritional Effect of NaCl on Shoot Growth ............................

11. Inhibition of Shoot Growth in Dicots and Monocots ..................................... A. The Timing of the Growth Inhibition ........................... B. Salinity Effects on Cell Extension .......................................................... C. Salinity Effects on Primordium Formation an ence ........... ............. D. Salinity Effects on Cell Division in Leaves ..

I I5 11s

121 121 123 123 12s

............................. 111. NaC1-induced Disruptions of Nutrient Transport . A. Influence of Some Experimental Conditions ................................. B. Effects on Whole Shoot Nutrient Accumulation. ...................................

126 126 128

IV. Nutrient Transport to Growing Shoot Tissue Under Salinity ......................... A. Protection of Growing Tissues ......................... B. Levels of Na and K in Young ...................... C. Disturbed Ca Status in Young .................................................... D. Other Nutrient Disruptions in E. Effects in Young Tissues Co F. Genotypic Salinity Effects i G. Lactuca sariva: a Model Dicot System ..... H. Summary: Salinized Nutrition of Growing Shoot Tissues .......

132 I33 133 137 138 139

V.

The Shoot Meristems: Special Nutrient Transport Challenges ...................... A. The Nutrition of Rapidly Dividing Cells: Possible Effects of Salinity .. B. Transport to Zones Proximal to the Meristem in Poaceae .....................

Advances in Botanical ReKidrch Vol. 29 incorporating Advances in Plan1 PdlhoIvpy ISBN 0-12-005920-0

141

141 143 146 148 149

Copyright 0 19W Academic Press All nghts of reproduction in any form reserved

114

D. B. LAZOF and N. BERNSTEIN

VI. Phloem Transport and Ion Recirculation under Salinity ................, ,.............. A. Remobilization of Nutrients from Ageing Shoot Tissues, ‘Long-term Recirculation’ ...,.......,.............................................................,....,. .......... B. XylemPhloem Transfer, ‘Short-term Recirculation’ C. Calcium Recirculation in the Shoot ........................................................ D. Summary of Salinization and Recirculation ........................

150

Nutrients ......................................

Nitrogen .................................................................................................. Micronutrients .............................................

157 157 157 158 158 160 162

Study of Nutrient Status and Transport on t Kinematic Growth Analysis and Elemental Microdissection .......................................... Specimen Preparation Considerations ........ Electron Probe X-ray Microanalysis .................................................... ... Secondary Ion Mass Spectrometry (SIMS) ............................................. Some Other Microanalytical Techniques ............................ ....................

162 163 166 166 167 168 170

VII.

Salinization and Shoot Nu

........

...........

B. Calcium ................................................................................................... C. Magnesium ..............................................................................................

E. F. VIII. The A. B. C. D. E. E

IX. Summary and Future Prospects A. Reassessment of Current St B. Model Systems ......... C. In Situ Elemental and sis ................................................. Acknowledgements ......................................................................................... References ............ ...............................................................................

151 154 155 156

171 171 173 174 175 175

The inhibition of shoot growth by NaCl salinization is reviewed from the perspective that determination of primary causes must involve evaluation of rapidly growing tissues specifically. Only within the minute volume of tissue comprising the zones of cell division and rapid cell extension can the direct causes of inhibited growth be found. Likewise, only there, can the events be identifed which allow the relevancy to be judged of more remote physiological changes. The hypothesis that a disturbance in mineral nutrition might be a primary cause of the NaC1-induced growth inhibition is evaluated within this framework. The review should be considered as an early evaluation of the hypothesis, given the paucity of data spec8ificallyrelevant to the minute zone of rapid growth and the similar paucity of anulyses for nutrients other than potassium, sodium and chloride. Data is reviewed und discussed which reflects on processes related to the maintenance of nutrient transport towards and into the meristem and rapidly extending cells, recognizing that this tissue is rather dissimilar to the whole, or mature shoot both in anatomy and transport properties. Recommendations are made fix the development of advanced methods of analysis towards the goal of quantibing alterations in nutrient transport and status within the minute zones of rapid growth.

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

I.

115

INTRODUCTION TO THE INHIBITION OF SHOOT GROWTH BY SALINITY

More than 80 countries suffer from water shortages each year (Gleick, 1993) and agriculture consumes more fresh water than any other human activity (Falkenmark, 1989). Regional water shortages are partially due to both the tendency of agriculture to degrade water quality and increasing agricultural demands (Pimentel et al., 1997). Salt-affected soils include about one third of the world’s irrigated soils presently and that portion is expanding (Downton, 1984; Chauhan, 1987). Diminishing supplies of fresh water for irrigated agriculture have increased competition for the resource and have led to abandonment of salinized agricultural land. A poor understanding of the mechanisms of the salt-induced inhibition of growth and of genotypic salt tolerance have hampered attempts to isolate specific genetic factors and develop new cultivars with improved salt tolerance (Cheeseman, 1988; Dracup, 1991; Munns, 1993). Crop scientists and farmers are interested in the development of new cultivars and in the modification of management practices in order to avoid the inhibition of growth. This growth inhibition is, during the first several days of stress, primarily restricted to the plant shoots (Munns et al., 1982; Munns and Termaat, 1986). A systematic approach in either of these endeavours requires an understanding of the underlying physiology. Despite the substantial genotypic variations in response to salinity which can be found in nature (e.g. Rozema et al., 1978; Venables and Wilkins, 1978; Cuartero et al., 1992) and among cultivars which were not intentionally bred for their salinity tolerance (Qureshi et al., 1980; Kingsbury and Epstein, 1984; Azhar and McNeilly, 1987; Ashraf and McNeilly, 1990; Taleisnik and Grunberg, 1994), it remains unclear what the primary physiological responses are which result in the inhibition of shoot growth and how these might best be determined (Munns, 1993). Provisionally, the simplistic answer might be that the primary responses can be identified by precise measurements at both the specific locale and time at which growth is affected. However, making precise measurements to show exactly when and where physiological processes occur account for most of the challenge in physiology. A.

GROWTH INHIBITIONS: GENERAL CONSIDERATIONS

Cell division and cell extension ultimately account for all shoot growth, although the tissue volumes in which they occur are usually merely a minute portion of the whole plant shoot. Where shoot growth inhibition occurs is usually quite some distance from the plant part which is directly exposed to salinization, namely the roots. So the concept of ‘primary response’ needs some qualification. For example, even if Na’ were to transport quickly to the growing tissue of the shoot, enter meristematic cells and inhibit a particular enzyme involved in cell division, the ‘primary response’ to salinization might be considered to be the increased transport of Na’ to the shoot apical meristem (SAM) and other processes in the plant might

116

D.B. LAZOF and N. BERNSTEIN

be affected before the “a] increases within the growing tissues. Still, the ‘immediate cause’ of the inhibited shoot growth would have to be found in the processes, regulatory events and metabolic changes occurring within these growing tissues. In this hypothetical example the primary and direct response of increased Na+ uptake and transport would bring about the immediate cause of the growth inhibition (the enzyme’s inhibition). However, the immediate cause may be more indirect. If, for example, photosynthesis immediately decreased by 90% upon salinization and remained at that level unless the plant were transferred to unsalinized medium, this would be an important response. However, to demonstrate that carbohydrate supply was critically limiting shoot growth it would still be necessary to show, at least, that transport of carbohydrate level to the growing tissues was affected following the photosynthesis effect. For either of these hypothetical cases, the crucial plant responses need to be discriminated from a number of organismal plant responses based on connection to the immediate cause within the growth zone of the shoot. The timing of the inhibition of shoot growth is as important to defining the mechanisms of the toxicity and of the tolerance-related response, as is the locale. Factors contributing to changes in cell division or cell extension (e.g. increased “a] or decreased carbohydrate supply) need to be established prior to the growth inhibition. Methods such as whole shoot growth analysis have on occasion demonstrated tendencies, at least, towards reduced growth within 1 to 2 days of initial salinization (e.g. Cheeseman and Wickens, 1986; Wickens and Cheeseman, 1988; Cramer et al., 1994b). However, it remains doubtful whether the precision in these methods allows evaluation of 1 day growth effects (see discussion in Wickens and Cheeseman, 1988). This doubt is founded not only on considerations of biological variability and the resulting statistical limitations, but also involves the necessity of bulking slowly growing and non-growing leaves (or, in the case of grasses bulking growing and non-growing portions of the same leaf) with the much smaller rapidly expanding leaves (or portions of leaves). Recent methodological developments such as the use of linear variable differential transformers (LVDTs) are discussed below with respect to their relevance in determination of immediate plant responses (section 1I.A). Reasonably, major physiological interest lies in those alterations which account for differential genotypic plant response. The increased Na’ accumulation or the reduced carbohydrate supply becomes much more interesting if they occur to a far greater extent in a species or a cultivar which are less tolerant of salinization, for in this case there are prospects for producing an improved cultivar. Experimental results bearing on genotypic differences are presented within most major sections of this review (sections I.C.3., III.B.3, 1V.F.) and summarized in section 1X.B. B. NaCI-INDUCED INHIBITION OF SHOOT GROWTH: GENERAL HYPOTHESES

Hypotheses for the mechanism by which salinity reduces shoot growth can be grouped into four general categories, each having several possible variations. One

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

I17

suggestion is that salinity reduces photosynthesis, which in turn limits the supply of carbohydrate needed for growth. A second is that salinity reduces shoot growth by reducing turgor in expanding tissues, which are not able to fully osmoregulate in response. A third is that roots sense salinity and down-regulate shoot growth via a long distance signal. Fourth, a disturbance in mineral supply to the shoot, either an excess (we have emphasized Na '- excess) or deficiency, might directly affect growth. The only hypothesis treated in great detail here is this last one. Each of the remaining hypotheses has some merit and may contribute in some way towards the long-term effects on growth. They are summarized briefly and can be explored more thoroughly in the reviews cited.

I . Disturbed Photosynthesis Photosynthesis may well be disturbed by salinization (e.g. Robinson et ul., 1983; Ye0 et al., 1985; Munns, 1993); however, it seems unlikely that reduced photosynthesis leads to reduced shoot growth. Increased starch accumulation has been reported for the whole shoot (Gauch and Eaton, 1942), mature leaves (Lechno et al., 1997) and for expanding leaf tissue (Rathert et af., 1981; Munns et al., 1982; Aslam et al., 1986) under salt stress. After weeks of salinization, photosynthetic activity per unit leaf surface area can be little affected by salinization, even though reduced photosynthetic leaf surface was exerting a large effect (Robinson et al., 1983; Kaiser, 1987; Munns, 1993; Cramer et al., 1994a), or even though the cells had accumulated high levels of salt (Fricke et al., 1996). In Zea mays, reductions in net photosynthesis did not occur during the first 5 h of salinization, even when the elongation of young leaves were inhibited in the same time frame (Cramer et al., 1994a). In Oryza sativa photosynthesis per leaf area did decline after 10 days of salinization, whereas the [NaILearsteadily climbed (Ye0 et al., 1985), but this might have been accounted for by accelerated leaf senescence (Ye0 and Flowers, 1989). In salt-sensitive Cirrus reficulata there was no reduction in net photosynthesis until 30 days after treatment with 50 mM NaCl (Walker et al., 1982). And in Triticum species there was actually an increase in I4CO2 fixation after 10 days at 2 5 m M NaCl even though net growth had been reduced by 20% (Passera and Albuzio, 1978). The inhibition of leaf growth, then, might be primarily responsible for reduced leaf area and a loss in photosynthetic capacity, not vice versa. Although primary carbon fixation is not a likely primary cause of reduced shoot growth, it remains possible that some other aspect of carbon utilization plays a more major role. A NaC1-induced disturbance in the supply of carbon to the growing zones of shoots might be associated with the increased starch accumulation in mature leaves. Further discussion of photosynthesis and salt stress is available elsewhere (Munns, 1993). 2. Reduced Turgor and IitsufJicient Osmoregulation The hypothesis that crop productivity may be limited during salt stress primarily by the requirement for increased turgor (e.g. Oertli, 1966, 1968; Flowers et al., 1991) and limitations of plant capacity for osmoregulation, has seen a great deal of

118

D.B. LAZOF and N. BERNSTEIN

criticism (Termaat et al., 1985; Munns and Termaat, 1986; Kramer, 1988; Munns, 1988). Turgor of growing tissues can remain unaltered in expanding leaves (Termaat et al., 1985; Cramer, 1992b), in the expanding regions of grass leaves (Matsuda and Riazi, 1981) and in whole shoots (Imamul Huq and Larher, 1984) experiencing a salt stress. Turgor may also return to near control levels after a transient decrease (Cramer and Bowman, 1991a; Arif and Tomos, 1995; Fricke, 1997). This is similar to the lack of enduring alteration in turgor found in plants experiencing water stress (e.g. Ackerson, 1981; Meyer and Boyer, 1981; Michelina and Boyer, 1982; Van Volkenburgh and Boyer, 1985). Indeed, an increase in turgor at the shoot apical meristem (SAM) has been indicated during water stress (Barlow et al., 1980). During the initial few minutes of exposing roots to a moderate salt stress, leaf expansion sharply decreases. However, such reductions seem to be largely restored within a few to several hours with adjustment of the cell wall’s yield threshold (Delane et al., 1982; Termaat et nl., 1985; Cramer and Bowman, 1991a; Cramer, 1992a). The reduction and recovery of leaf expansion may be coincident with the loss and recovery of cell turgor in the expanding leaves (Cramer and Bowman, 1991a; Cramer et al., 1994a). Where changes in turgor have been shown after transfer to salinized medium these have also been rapid, transient and reversible within 30 min of transfer (Fricke, 1997). Elsewhere, decreases in turgor of expanding leaves have been correlated with the initial changes in leaf expansion rate (Wilson et al., 1970b; Cramer, 1992a; Munns, 1993). These rapidly occurring changes in leaf expansion rate (minutes) were similar for salt sensitive and salt tolerant genotypes and so probably are not relevant to salt tolerance mechanisms (Wilson et al., 1970b; Cramer, 1992a). Several years ago Greenway and Munns ( 1980) suggested that osmoregulation was probably of little importance in the extent to which a genotype can cope with salinity stress, since virtually all plants seem able to osmoregulate, regardless of their relative sensitivity. Recently, it has been shown that in one case a single gene mutant with 20-fold increased sensitivity to NaCl actually accumulates 80% more proline than the wild type (Liu and Zhu, 1997). Results supporting an important role for cell turgor in salt-induced reductions of leaf expansion have been rare (Neumann, 1997). 3. A Signal from the Roots Several reports have suggested that a signal from the roots communicates with the expanding leaves and growing tissues of the shoot (e.g. Termat el al., 1985; Munns and Termaat, 1986; Rengel, 1992) and that this may be a similar process in water stress (Zhang and Davies, 1990; Ball and Munns, 1992). There is a good deal of circumstantial evidence that abscisic acid (ABA) may be involved in regulating the shoot response at the shoot (Munns and Cramer, 1996). For example, spraying ABA on the shoot of Lens culinarus partially ameliorated (74% recovery) the NaC1-induced inhibition of shoot growth (Bano and Hayat, 1995). However, it is unlikely that the levels of ABA transported to the shoot control the level in the growing tissues (Munns and Cramer, 1996). Although there is much circumstantial evidence that ABA is somehow involved, the long distance signal remains a

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

119

mystery and there has been no progress in identifying any other factor which might be transported to the shoot and control ABA synthesis, turnover or compartmentation in growing shoot tissue (Munns and Cramer, 1996). The 'root signal hypothesises' remains to be thoroughly explored and tested. There is a limited similarity between this hypothesis and that of disturbed nutrition, as nutrients themselves might be considered long-distance messengers. After all, many nutrients have an essential role in the processes of cell division and cell extension and these would cease soon after the supply were halted, especially in tissues with little nutrient storage capacity. C.

A NUTRITIONAL EFFECT OF NaCl ON SHOOT GROWTH

Within the general hypothesis of a NaC1-induced disturbed nutrition, the dominant specific hypothesis has clearly been that of 'ion excess', i.e. the idea that Na' and/or C1 rise to toxic levels in the shoot, eventually to high levels in the cytoplasm leading directly to metabolic inhibitions. In a widely invoked version, the selectivity for K+ over Na becomes increasingly compromised with the severity of salinization and this leads to failure in maintaining an adequate [K] in the cytoplasm (e.g. Abel and MacKenzie, 1964; Lauchli and Wieneke, 1979; Flowers and Yeo, 1981; Jeschke, 1984). 1. Ion Excess and Selectivity Hypotheses Most nutritional physiological studies have assumed the idea of Na:K competition at the onset, by only considering Na' effects on K + transport and nutrition alone. In surveying 50 research articles published between 1980 and 1995 it was found that accumulation or transport data for Na' or C1, transport or accumulation of K+ was studied in 72%, whereas only 18% studied transport or accumulation of a nutrient other than K'. All reports which included transport of nutrients other than K', included K' as well and in most (60%) of these the focus was clearly on K+ effects. Besides the fact that salt-induced effects on other nutrients have rarely been considered, the idea that selectivity for K' over Na' in the shoot is of primary or overwhelming importance in salt tolerance has been carefully criticized (e.g. Munns et al., 1982; Cheeseman, 1988). One questionable aspect is the idea that high [Na] in the cytoplasm underlies the growth inhibition. However, high cytoplasmic and chloroplastic [Na] did not reduce photosynthesis per unit leaf area (Robinson et al., 1983; Schachtman et a/., 1989). Furthermore, high cytoplasmic [Na] has been reported for halophytes (Harvey et al., 1981), without any evidence that these have specially adapted enzymes (Flowers, 1972; Greenway and Osmond, 1972; Flowers et al., 1977; Yeo, 1981; Hajibagheri et al., 1985). Indeed, cytoplasmic enzymes of halophytes seem to be similarly sensitive to NaCl in vitro (Flowers et al., 1977), whereas cell wall enzymes from neither halophytes nor glycophytes show much salt inhibition (Banuelos et al., 1996). Evidence that Na' and C1 are more poorly compartmentalized in glycophytic shoot tissue remains tenuous (section VIII).

120

D. B. LAZOF and N. BERNSTEIN

2. Shoot N u f Accumulation: a Disadvantage? Although not an essential nutrient for all plants, Na’ is probably accumulated advantageously in the shoots of all plants under some conditions as an inexpensive omoticant (Yeo, 1983). Indeed, halophytes generally accumulate higher Na’ levels in their leaves, than do glycophytes (Flowers et al., 1977). This obvious challenge to the ‘ion excess hypothesis’, has been overcome, by inventing two classes of plant with respect to salinity response, ‘Na includers’ and ‘Na excluders’. However, even in the most salt-sensitive glycophytic genotypes, Na’ when applied at low levels (ca. 10% of levels causing 50% growth inhibition) has often stimulated shoot growth (e.g. Elzam and Epstein, 1969; Marschner et al., 1981a,b; Lazof and Cheeseman, 1988b). If researchers were to avoid the simplification of ‘excluder’ and ‘includer’ species, then perhaps, a much deeper criticism of ‘ion excess’ might follow (Cheeseman, 1988). Common physiological limitations to growth encountered by both halophytes and glycophytes with respect to salinization might emerge. Indeed there has been a great deal of study of physiological response in halophytes at growth-inhibiting levels of NaCl and there is no reason, a priori, for excluding these from a discussion of salt-induced growth inhibition. Indeed these are probably more enlightening than studies of halophytes at stimulating NaCl concentrations for comparison to the glycophytic growth inhibition. True, many halophytes possess specialized structures for compartmentation and secretion of salt, but not all halophytes possess such specialized structures. Those that do not, although mislabelled ‘pseudo halophytes’ by some (Breckle, 1995), might well be the more productively investigated group due to their lack of obvious morphological disparity with salt-sensitive glycophytes (Cheeseman et al., 1985). 3. N u f Exclusion and Genotypic Tolerance Several investigators have indicated that Na’ exclusion from the shoot might be correlated to genotypic tolerance, including studies in Z. mays, Glycine rnax and Triticum X Lophopyrum derivatives (Lauchli and Wieneke, 1979; Hajibagheri et al., 1987; Schachtman et al., 1989, respectively). However, other studies have shown a lack of clear correlation between shoot Naf accumulation and genotypic tolerance (e.g. Lessani and Marschner, 1978; Rush and Epstein, 1981b; van Steveninck et ul., 1982; Walker et al., 1982; Grattan and Maas, 1988; Ashraf el al., 1990; Alberico, 1993; Reimann, 1993; Botella et al., 1997; Davenport et al., 1997; Leidi and Sairz, 1997). Due to the tremendous diversity in how such correlations have been pursued, the general applicability of this model remains in doubt. In G. max although there was a great difference between genotypes in [Na],hooland [Cl],h,ol at high salinity (100 mM NaCl), the more sensitive cultivar was already fully growth inhibited (and the tolerant not at all) at just 10 mM NaCl. And at this or [K]~hoolof the two low level of salinity there was no difference in genotypes (Lauchli and Wieneke, 1979). Similarly for one genotype of Lnctuca sativa grown with either 0, 1, 10, 50, 100 or 150 mM NaCl, growth was inhibited first at 50mM (14%) and then much further (41 and 66%) at 100 and 150 mM NaC1, whereas [K]\hooldecreased 47% at 5 0 m M and then not further at the two

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

121

higher salinities (Lazof and Cheeseman, 1988b). A more sophisticated formulation of the same general hypothesis might recognize that it would be nutritional disturbances within the growing zones in particular which may inhibit either cell extension or cell division. Owing to the paucity of existing literature relating to the condition of the zones of rapid cell division and extension subsequent to salinization, the existing literature relevant to the status of young (still growing) leaves and tissues is included in the following sections. First, a few additional details must be provided concerning the timing of NaCl induced shoot growth inhibition.

II. INHIBITION OF SHOOT GROWTH IN DICOTS AND MONOCOTS A.

THE TIMING OF GROWTH INHIBITION

The effect of salinization on leaf elongation is astonishingly rapid (0.1-2 h), under some conditions requiring no more than a few minutes after exposure of roots to salinized medium (Matsuda and Riazi, 1981; Cramer and Bowman, 1991a.b; Cramer, 1992a). This rapid response may, however, also be transient (Matsuda and Riazi, 1981; Delane et al., 1982; Cramer and Bowman, 1991a,b, 1994a) and rapidly reversible (Munns et al., 1982; Rawson and Munns, 1984; Cramer, 1992a). The elongation of Z. mays leaves decreased 65-85% within 2 min of adding NaCl to the external solution (Cramer and Bowman, 1991a). The leaf elongation rate (LER) readjusted within 1-35 min depending on the level of salinity. When the plants had been suddenly salinized to 80 mM NaCl or more the LER was reduced 25% after adjustment. Immediate restoration of LERs was reported following transfer of 2. mays from salinized media, even when salinized to 75 mM NaCl (Cramer, 1992a). The rapid decrease in LER may also be unrelated to genotypic sensitivity (Cramer, 1992a). although after longer adjustment to salinized medium the response correlated with the supposed genotypic sensitivities (5 h, Cramer et nl., 1994a). A varying capacity for adjustment and resumption of normal leaf elongation in three barley cultivars was reported using time-lapse photography, although their relative salt sensitivity was not established (Matsuda and Riazi, 1981). Similar to the subsequent work in Z. mays, Hordeum leaves ceased to elongate within 15 min of salinization to 9 or 1 1 bars of NaCl, reinitiating growth after 60-90 min. If the most rapid growth responses are both unrelated to sustained growth inhibition and to genotypic tolerance, then they may only mislead in the quest for the mechanism of the growth inhibition. The measurement of LERs by the LVDT method need not be restricted to the short term ( < 2 h). When a constant and sufficient driving force is provided as tension on the elongating leaf, larger reductions in LER with larger non-reversing components can be measured (Cramer and Bowman, 1991a). In 2. mays LERs were measured within 5 h of salinization which were in agreement with dry weight accumulation (2-7 weeks, Cramer and Bowman, 1991a; Cramer et al., 1994a). After a 5 h adjustment period the LERs of plants exposed to 80 mM NaCl remained

122

D. B. LAZOF and N. BERNSTEIN

Regulation of Growth-related Shoot Nutrition

r

Fig. 1. Flow diagram depicting the control over nutrient supply to the zones of shoot growth. Selected physiological effects and interactions, which could directly or indirectly affect shoot growth during salinization over the long term (several days to a few weeks, or more). Arrows in grey represent interactions (arrows cross behind other processes and parameters). A few processes occurring between blocks of the diagram are written adjacent to the blocks.

20-30% less than control rates, an effect similar to that for net 3 day leaf growth. Medium-term responses to salinity measured by LVDT have also appeared to be genotypically differential and in accord with NaCl sensitivity (Cramer et al., 1994a). The problem with studying physiological processes after several days or weeks of salinization is that initial responses trigger regulatory processes throughout the plant (Fig. 1). These processes occur at nearly every level of development and within each organ, each interacts and influences other and distant processes. The

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

123

task of unravelling which events lead to the non-adjusting, genotypically differential plant responses becomes increasingly difficult as the time frame extends, because changes in metabolic rates, depletions and surplus of various resources are present within each component, while adjustments to reduced growth influence metabolism throughout the plant (e.g. Greenway and Munns, 1980). The time frame worthy of the most intensive research is probably that in which genotypically differential responses are seen to arise first (i.e. 3 h) and continuing to a time at which consistency to longer exposures in extent of stress and genotypic sensitivity can be evaluated.

B. SALINITY EFFECTS ON CELL EXTENSION

Rapid (3-24 h) measurable effects on growth are always effects on cell extension, since dividing cells do not contribute significantly to net leaf expansion until several cell cycles (20-25 h per cycle, Clarkson, 1969; Powell et al., 1986). This does not preclude important effects on cell division also, only that the latter could not be detectable so quickly by macro methods. Following this reasoning, there can be little doubt that NaCl stress results in an inhibition of cell extension. Indeed, more than 25 years ago a decrease in the rate of biomass accumulation of Gfycine species was reported after as little as 1 day salinization (Wilson et al., 1970b). By measuring the mass of very young leaves (those first apparent to the unaided eye following salinization) were reduced 70-75%, even though still growing but older leaves showed no decrease after 3 days of exposure. Additional reports of effects on LER within the 3 h to 3 days time frame were discussed above (section 1I.A). After an 8 day salinization to 80mM NaCl cell length of Hordeum vulgare leaves had decreased 30% compared to the 1 mM NaCl control (Lynch et al., 1988). At present very little is known about the biochemistry or cell biology of the inhibition of cell extension in leaves subsequent to salinization. As for the biophysics, apparently the effects on cell extension in the relevant time frame are exerted by changes in the yield threshold of the cell wall and not by turgor, since osmotic adjustment can be complete and rapid within the zone of rapid cell extension (section I.B.2, Cramer, 1992a). Experiments within a longer time frame suggest that ABA levels might regulate cell extension during salt stress (e.g. Ban0 and Hayat, 1995), but apparently no experiments have yet been carried out measuring specific levels within the most rapidly expanding leaf zones.

C. SALINITY EFFECTS ON PRIMORDIUM FORMATION AND LEAF EMERGENCE

Whereas leaf emergence rates can be precisely determined, the point at which a leaf is said to emerge is arbitrary and a matter of convenience. Often for dicots it is simply taken as the smallest leaf observable to the unaided eye. In Poaceae the presence of a leaf tip beyond the whorl of older leaf sheaths is most often

124

D. B. LAZOF and N. BERNSTEIN

considered the point of emergence. This is also the point of transition from heterotrophic to autotrophic tissue. A plastochron index can be developed in monocots, as in dicots, by applying an arbitrary length beyond the whorl as the point of emergence. Leaf initiation in dicots, as opposed to emergence, is limited to the SAM. It involves rates and patterns of cell division (Hay and Kemp, 1990). Immediate effects on dicot leaf initiation would also be located within the meristem, where the primordia form, or at the step when primordia launch into intensive cell extension. In grasses 6 to 7 leaf primordia have often already formed in the seed, so that treatments imposed on the seedling could not affect formation of the first leaves. The rate of leaves passing through the emergence point might be affected differentially from a more strictly defined rate of leaf initiation based on primordia formation. Primordia might accumulate on the apex of a salinized plant with a delay in cells entering rapid cell expansion. Such details of leaf initiation under salinity have never been reported, however such accumulation did not occur during cold stress of wheat (Hay and Kemp, 1990). Indirect evidence suggests that salinity may affect leaf development close to initiation. Expansion of Sorghum bicolor leaves was found to be more strongly inhibited for leaves still enclosed in the sheath than for visible leaves (Bernstein et al., 1993b). Similarly, in dicots the greatest effects on ultimate leaf development were found in leaves not yet in the phase of rapid expansion at the time of salinization (Rawson and Munns, 1984; Aslam et al., 1986; Lazof et al., 1991). Ultimate leaf formation (undefined developmental timing) was reduced 35% in Phaseolus vulgaris and for both leaves and flowers by 50% (salinization to 150 and 70 mM NaCl, respectively Lagerwerff and Eagle, 1961; Hussain and Ilahi, 1995). With the exception of a study in highly salt-tolerant Beta vulgaris (leaf fresh weight was not reduced by the treatment), leaf emergence rate has consistently been shown to be particularly sensitive to salinity (Papp et al., 1983). In Atriplex amnicola the numbers of leaves emerging per day decreased continuously with the severity of the salt stress from 20% to 68% to 40% at 200,400 and 600 mM NaCl respectively (Aslam et al., 1986). In L. sativa leaf emergence was 6% slower in salinized plants and this appeared to be effective after just 6 days of treatment (Lazof et al., 1991). Similar decreases were found for salt stressed Hibiscus cannubinus during 4 weeks of salinization (Curtis and Lauchli, 1985). In H. vulgarr leaf emergence was also delayed 5-6 days for leaves 17 days after stepwise salinization was initiated (Rawson et al., 1988), or delayed 4-5 days after plants had been salinized for 20 days (Jeschke and Wolf, 1985). In four Glycine species the total number of leaves was reduced by ca. 50% in the more salt-sensitive species (Wilson, 1967). In salt-stressed S. bicolor the plastochron index of shoot development (leaf emergence per day) decreased by half relative to that of the non-salinized control plants during the first 5 days of salinization (Bernstein et d., 1993a). In particular, leaf number four was delayed in development, a leaf which was still unemerged from the whorl but rapidly elongating at the time of salinization (Bernstein et al., 1993b). After 23 days of salinization 0. sativa leaf

THE NaCl INDUCED INHLBITION OF SHOOT GROWTH

125

emergence was 2 days delayed specifically in the more salt-sensitive genotypes (Ye0 et al., 1991). Reduced whole shoot biomass of salt-stressed plants may result, in part, from either shorter periods or lower rates of leaf expansion (along with possible effects on stem growth). Although shorter periods and lower rates of leaf expansion are likely to be due to some complex effect of both cell division and cell extension, delayed primordia formation most likely represent effects on cell division specifically. It is thought that a critical number of cells needs to be formed within each leaf primordium before leaf initiation (Poethig and Sussex, 1985a,b; Dale, 1988). Microscopy has indicated that there is little cell expansion within leaf primordia (Esau, 1977; Lazof and Lauchli, 1991b), again suggesting that primordia form largely on the basis of cell numbers. The dominance of cell division during primordia formation is also suggested by 50-fold increases in the length of very young leaves coincident with increases in cell length of merely three- or four-fold (Sunderland and Brown, 1956; Dale, 1988). D. SALINITY EFFECTS ON CELL DIVISION IN LEAVES

In dicot leaves cell division continues, to some extent, up until 95% of the final leaf size is attained (Maksymowych, 1973; Dale, 1988) occurring largely within pockets of cells surrounding minor veins (Sachs, 1989). Even though some cell division continues through much of leaf expansion, the rate of cell production falls exponentially arriving at a low limit by the middle of rapid leaf expansion (Maksymowych, 1973; Lamoreaux and Chaney, 1978; Sachs, 1989). In Lactucu sativn, leaves gained fresh weight exponentially from emergence until they reached 540mg fresh weight but continued expansion for another week at less than one third the earlier rate, finally reaching about 3.Og (Lazof and Bernstein, unpublished). If the steady exponential expansion rate, as determined during the first few days of visible growth, were continuous from the instant at which primordia ‘launch’ into intensive cell extension (and exponential leaf expansion), then it would require about 1 day for primordia to reach the easily visible 5 0 m g fresh weight (FW). If the leaf development pattern in L. sativa is consistent with the general pattern in dicots (Maksymowych, 1973; Dale, 1988), then effects detected before reaching 270mg FW, would have occurred during a period in which cell division dominated leaf development. Ultimate cell numbers in grass leaves were significantly reduced by salinization (Munns and Termaat, 1986). Two studies, one in H . vufgure and one in I? vulgaris measured interstomatal distance, as an indication of cell expansion and judged that there was a major effect on cell extension (Brouwer, 1963; Munns et al., 1982). Such methodology is inconclusive, however, since a reduced rate of meristematic activity would not only affect the final number of cells per leaf, but could also (or exclusively) affect the number of primordia formed and leaves initiated. It is uncertain also whether final epidermal cell size reflects the size of underlying mesophyll cells, which account for most leaf cells. were studied during salinization and Leaf expansion rates in Heliunfhus ~~rznuus

126

D. B. LAZOF and N. BERNSTEIN

during either the 0-24 or 24-96 h period after removal of plants from salinized medium (Rawson and Munns, 1984). Leaf expansion rates increased (on average for all leaves) 15%and 59%over the rates in saline solution, during the initial 24 h after removal from media salinized to 50 or 100mM NaCI, respectively. The expansion rates then decreased 21% and 28% from that initial 24 h rate during the next three days. The leaf expansion rates in salinized plants was increased over that of control plants by 10% and 8%, suggesting that cell extension was repressed during salinization and derepressed during the initial recovery (see section 1I.B). Although effects on cell extension may have been generalized, acting on all expanding tissues within the shoot, younger leaves (numbers 14 to 19) had the most severe reduction in expansion rate during salinization, whereas older leaves (numbers 8 to 14) had the largest increase in rate during the recovery period. In the younger group of leaves (or fully formed primordia) the pool of cells ready for rapid cell extension, then, either had not increased over the control level during the 10 day period of salinization, or they were present in primordia which were repressed from launching into intensive cell extension. This young group of leaves was barely or not yet emerged at the time of initial salinization (just barely measurable with a ruler, personal communication R. Munns). Hence, in these leaves cell division was still a dominant process during salinization. If the repression of cell extension is indeed general throughout expanding shoot tissues, then the younger leaves in showing the largest irreversible growth inhibition suggests a strong effect on cell division specifically, since cell division was the dominant process during the treatment. Furthermore, if cell division had not been inhibited, there would have been a higher potential for increased expansion in these younger leaves upon derepression. Some biochemical work has also had a bearing on effects of salinization on cell division. Protein synthesis was reduced in Nicotiana tabacum by 50% after 20 h of salinization (Ben-Zioni et al., 1967). This reduction was only slightly greater in young leaves (not defined specifically) than in older leaves (57 vs. 48%reduction). Rapid effects of drought or salt stress on polyribosome formation in the whole shoot might also be taken as circumstantial evidence that protein synthesis may be inhibited crucially in meristematic cells, where rates of protein synthesis are most intensive (Rhodes and Matsuda, 1976). Decreases in root tip protein synthesis upon salinization were found for Pisum sativum and Glycine m x (Rauser and Hanson, 1966; Kahane and Poljakoff-Mayber, 1968).

111. NaCI-INDUCED DISRUPTIONS OF NUTRIENT TRANSPORT A. INFLUENCES OF SOME EXPERIMENTAL CONDITIONS

Complex and variable plantlmicrobe, /soil, and /climate interactions are the norm for most environments in which plants grow. Simplification, however, is necessary to isolate factors and observe the resultant responses. The experimentalist typically

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

127

attempts to restrict the extent of departure from common natural conditions. A compromise between these inherently conflicting objectives is usually sought. However, enhanced statistical significance can be achieved by employing overly simple, unrealistic systems. This becomes especially troubling when extreme conditions are not properly discussed within a report. Two such cases are of special importance to salinity research. First, more often than not, salinized plant response has been compared to that of plants growing in the absence of Nat (nominal 'absence', actually an undetermined micromolar level). Second, much experimentation has considered effects of salinity with enormous Na to Ca molar ratios in the treatment solution. The critiques and implications of these two design flaws are discussed below. Several additional aspects of experimental design, however, although important in understanding the salinity response, are not discussed nor involved in the remaining chapter. These include, most notably, salinity effects of salts other than NaCl (e.g. Na2S0,), specific chloride effects and effects on the nutrition of plant roots. I . Control Levels of Na Low levels of Na' have been found to be growth-stimulating to some glycophytes (section I.C.2). This could possibly prove valid for a wide range of species. In all cases the plants accumulated significant Na' at growth stimulatory levels. Metabolism is undoubtedly altered at these levels of exposure, at the very least growth-related metabolism. It is therefore completely incorrect to ascribe all metabolic alterations at inhibitory levels of Na' to deleterious NaCl effects without examination of plant status under conditions of Na-induced growth stimulation. For example, consider that the Na:K ratio in treatment solutions can be varied over an enormous range while maintaining Na' well below inhibitory levels (Marschner er al., 1981a). Growth was stimulated by the increased Na' levels which never reached 5 m M (inhibition commences at levels close to 100 mM, Marschner et al., 1981b). Not only have less salt-tolerant crops shown growth stimulation by low levels of NaC1, but also in monocots increased Na:K ratio of the nutrient medium gave rise to more than a doubling in shoot tissue Na:K even though shoot growth inhibitions were either nil or 50% (Z. mays, Botella et al., 1997). Increased Na and decreasing levels of the major mineral osmoticant (K) are logical consequences of increasing [NaImedlum, independent of whether growth is inhibited. The importance of the effect of salinization on (NaIrhoo,and [K]shoo,has been artefactually exaggerated by the practice of using a '0 Na ' ' control treatment. This conclusion is further supported by hundreds of reports on Naf uptake and translocation in halophytes, many of which have been conducted at growth stimulating levels of NaCl (e.g. Flowers er al., 1977; Lauchli. 1986; Breckle, 1995).

2. Na:Cu Ratios There is nothing new about the concept that the Na:Ca ratio of the soil solution affects the extent of NaCl stress. The ability of increased soil Ca to protect plants from salinity stress was reported almost 100 years ago (Kearney and Cameron,

128

D.9.LAZOF and N. BERNSTEIN

1902; Kearney and Harter, 1907). Sodium absorption ratios (SARs), where SAR = Na/[(Ca

+ Mg)/2Ios (elements in mequivA)

are specific mineral forms of the Kerr Gapon equation for the equilibrium between cations in solution and held on a colloidal matrix. Explicit warnings have appeared previously against conducting salinity studies at unrealistic SARs (Maas and Grieve, 1987) with plant growth inhibition being heavily dependent on the solution Na:Ca ratio (e.g. Grieve and Fujiyama, 1987; Grieve and Maas, 1988; Gorham and Bridges, 1995). Ratios of Na':Ca2+ > 100 are rare in nature and seawater has an SAR of about 85. A 0.25X strength Hoagland solution (1 and 0.25 mM in Ca and Mg, respectively) would need to approach 170mM in Na' to reach an SAR of 150. Irrigation waters with SARs in the 20s have been classed as 'unsatisfactory for use in imgation', since much higher SARs result as the soil begins to dry (Hawkes et ul., 1975). Most physiological salinity work has been conducted within a more realistic range for salt-sensitive plants using Na:Ca ratios < 80, and/or SARs < 150. The following sections of this review have been based almost entirely on studies, or on treatments within such a range. Although any such limits are somewhat arbitrary, establishing some liberal limits allows a more meaningful interpretation of the literature. Explicit comments on Na:Ca ratios and SARs have also been included for many of the studies below to this same end. Where the nature of the treatment has been properly discussed and the results interpreted within the framework of their relevance for natural environments, some studies with extreme Na:Ca ratios have been retained, often with qualification of the experimental conditions. It might also be noted that effects of salinization at constant Na:Ca ratios have been studied and that growth inhibitions do occur under these conditions (e.g. Eaton, 1942; Lagerwerff and Eagle, 1961; Abel and MacKenzie, 1964; Pitman, 1965; Bernstein et al., 1969; Shannon, 1978; Ehret et L J ~ . , 1990). The major emphasis of the present review is phenomena within growing shoot tissues. Although there seems little reason for assuming that increasing or decreasing [ K]ahoothave anything to do with growth inhibitions or with a particular genotype's ability to cope with salinity (section I.C.l), there may be value in evaluating the evidence for inhibitions of nutrient supply to the whole shoot (sections 111 and VII). It is undeniable that all nutrients which are supplied to growing shoot tissues are subject first to the control of general translocation to the shoot. Furthermore, the discussion of whole shoot nutrition provides a framework for discussion of the less extensive literature available on the nutrition of growing shoot tissues under salinity. B. EFFECTS ON WHOLE SHOOT NUTRIENT ACCUMULATION

There are several points in the pathway of nutrient supply to growing shoot tissue, where transport might be disrupted, including: root uptake, radial transport, transendodermal transport, xylem loading, long-distance transport, reabsorption by xylem parenchyma, partitioning within the shoot, whole shoot retranslocation.

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

129

inter-leaf retranslocation and leaching through leaf cuticles. Most of the work on disturbed transport to the shoot has followed the suggestion that salt tolerance in glycophytes would be evidenced as lower shoot accumulation rates for Na', with the frequent inclusion of the hypothesis that K + shoot accumulation rates would also be better maintained. These studies have shown universally that Na' accumulation in the shoot is much greater and K' accumulation much decreased under conditions of high NaCI. This finding is neither surprising nor in dispute (section 1.C). nor are the important metabolic roles of K + in doubt. However, these roles may be fully preserved at relatively low [KIshoot(section I.C.1). More important questions for the hypothesis of disturbed nutrition might be: (1) whether the level of K + becomes so low as to inhibit growth or metabolism; ( 2 ) whether any similar disruptions in mineral nutrient supply occur for other nutrients; and (3) whether any such disruptions correspond to the locale and time frame of well documented growth inhibitions. Calcium is a particularly interesting nutrient for evaluating these questions, considering that it is known to be both essential and closely regulated during the processes of both cell division and extension. It has also been studied more extensively with regard to salinity than any other nutrient excluding Na' , CI- and K'. 1. Translocation of Ca2' to the Shoot: Comparison with K' Translocation of Ca" to the shoot seems to be more drastically affected by salinization than is the root accumulation of Ca2+ and often even more reduced than that of K + . In Gossypium hirsutum [CaIshoo,decreased 60% more than [Ca]rool (Na:Ca of 20, Kent and Lauchli, 1985). In an unimproved line of Trifolium prutense the [CalShoo,decreased almost 60% with salinization to 100 mM NaCI, while [Ca],,,,, was unchanged with similar results for unimproved lines of both Medicago sativa and Trifolium alexandrinum at moderate SARs (Ashraf et al., 1986). In L. sativa [CaIshootwas decreased 67% but [CaIrootonly 40% (SAR of 113, Cramer and Spurr. 1986). In a salt-sensitive cultivar of Hordeum vulgare 32 h root accumulation of Ca2+decreased 11% whereas shoot accumulation decreased 55% (Lynch and Lauchli, 1985). Also in H. vulgare exposed to 75 mM NaCl for 7 days, the 4 h root accumulation of 4sCa2+was decreased by 63%, while the translocation to the shoot was reduced 94% (Na:Ca > 150 and SAR > 140, Cramer et al., 1989). Although this latter salinity treatment was rather extreme, greater shoot than root reductions (56 vs. 30%) were found with a more moderate salinity treatment also (Na:Ca of 7.5 and SAR < 70). In two lines of each of four grass species [Ca]roolwas decreased very little, whereas the [Callearwas reduced 60-85% in all four species at the most severe salinities (Ashraf er al., 1990). In a relatively salt-sensitive wheatgrass, Agropwon intermedium, [CaIshootdecreased twice as much as did [Ca]roolwhen salinized to 20 mM NaCl (Elzam and Epstein, 1969). In salt-stressed G. hirsuturn [Ca]shooldecreased significantly more than [KIshw, (87 vs. 57%. for a NdCa of 20, Kent and Lauchli, 1985). Although this may have been partially due to luxury consumption of Ca2+ in the '0 Na' solution with l0mM Ca, even when compared to the I mM Ca control, the [CaIbhoo, had been

130

D. B. LAZOF and N. BERNSTEIN

reduced 83% by 200mM NaCl. In G. mux a 27% decrease in [CaIshoorwas accompanied by a 30% increase in [KIshoo,at increased salinities (Na:Ca< 16, Grattan and Maas, 1988). In 7: pratense (the most salt-sensitive species of the three tested) [Ca]shooland [KIshootdecreased 56 and 14%, respectively (Ashraf et al., 1986). In L. sativa [Ca]sl,oo,decreased slightly more than [KIshoot(Cramer and Spun-, 1986). In salt-stressed Persea americnna [Ca],,,, and [KIlcf decreased 65% and 8%, respectively, in the most salt-sensitive genotype (Downton, 1978). In Phaseolus vulgaris [Ca]shootdecreased 46% whereas [KIshoolincreased 38% after salinization for 21 days ( I mM Ca, Kawasaki and Moritsugu, 1978b). In Lupinus luteus (only two spectra shown) the vacuolar [Ca] of the spongy mesophyll decreased by more than half with no decrease in the K level (van Steveninck et al., 1982). Even in the case of salt-stressed halophytes, well adapted to osmotic replacement of K+ with Na+, the [Call,,- has sometimes decreased much more than the [KIleaf(88% vs. 76%, Ye0 and Flowers, 1986). As for monocots, in moderately salt-stressed Zea mays [Ca],,,, was decreased 54% and [K],,,, not at all after 8 weeks (Benes et al., 1996). The [CaIshoo,of a relatively salt-sensitive Zeu muys cultivar decreased 12-fold more than did the [K]slloo,6 or 18 days after salinization (Na:Ca = 80, Cramer et al., 1994b) and nine-fold more than the decrease in [K]ahool (Na:Ca = 40, Kawasaki and Moritsugu, 1978b). In H. vulgare [CaIshool was reduced 49% more than [K]shoo,after 21 days of salinization, although in 0.sativa [Ca]sl,ool increased while [K]shooldecreased 60% (Kawasaki and Moritsugu, 1978a). In the four grass species Holcus lanatus, Lolium perenne, Dactylis glumerata and Festucu rubra reductions in [CaIshootwere greater than those for [KIshm, (ca. 600, 400, 50 and 50% greater reductions, Ashraf et ul., 1990). In salt-sensitive Agropyrun decreased 87% and [K]ehoo, not at all with a salinization intermedium the [Ca]sl,oot which reduced growth 90% (low Na:Ca, Elzam and Epstein, 1969). Although, NaC1-induced K deficiency has received intense study, it is rare that [CaIshmtand [KIshoot were both determined and [KIshooldecreased more than [CaIshoot.In the more sensitive of two corn cultivars the decrease in [KIshool exceeded that of [CalShoat and occurred only in one of the two treatments (100 mM NaC1, Fortmeier, 1995). In H. vulgare the [CaIshootdecreased 25%, while the [K]nhootdecreased 45%, but the salinized treatment solution had been increased fourfold for Ca and not at all for K (Gauch and Eaton, 1942). Perhaps, the weak reductions of [CaIshootin these two reports were, in part, due to the rather low Na:Ca ratio and SAR (13 and 14, respectively). The reductions in [K]shoor exceeded the reductions in [CaIshootin just two of seven crops after salinization for 23 days to 100 mM NaCl (Lepidium sativum and Capsicum annum, Lessani and Marschner, 1978). In 0. sativa while the Na:Ca ratio of the medium was directly conelated with the whole shoot Na:Ca ratios, neither of these were correlated with the relative inhibition of shoot growth at moderate SARs (Ye0 and Flowers, 1985). 2. Ca2+translocation: comparison to A@+ Calcium and magnesium are divalent, cationic, essential nutrients. They both typically accumulate in shoot tissue at the level of a few to several pmol (g fresh

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

131

weight)-’. One important difference is that whereas Ca2’ is widely considered to be phloem immobile and incapable of transport symplastically, Mg2+ is thought not to be similarly restricted. In 7: pmtense [Cali,,,. was decreased 34% on average with the greatest reduction for the most salt-sensitive genotype, whereas decreases in [MgIleaf were about 20% with little variation (Ashraf et al., 1986). In L. sativa salinized for 20 days to 120 mM NaCl the reduction in [CaIshoo, was ca. 25% greater than was the reduction for [Mglsh0,,,(Cramer and Spun, 1986). In salt-stressed F! americana [CaJleafdecreased 65%, whereas [Mg],, decreased just 23% in the most saltsensitive F! americarza tested (Downton, 1978). In Lupinus albus the [Mg] of the xylem was increased less than was the [Ca] (2.8-fold vs. 3.8-f0ld), and the petiolar [Mglphloem was reduced only half as much a [Ca]phlcKln (Jeschke et al., 1986). In P. vulgaris [Ca]shoordecreased 46% after salinization for 21 days, whereas [MgIshoo, decreased only 20% (Kawasaki and Moritsugu, 1978b). The comparison of changes i n [Ca] and [Mg] in shoot tissues appears to be much the same for monocots. The [Cali,,, in Triticuin aestivum and H . vulgare at a Na:Ca of 5 and an SAR < 8.5 were decreased 49% and 72%, respectively, after 3 weeks of salinization, despite the fact that [Ca]mrd,um was 2.6 times greater in the salinized treatment (Ehret et al., 1990). The [MgIledincreased 3.5-fold and two-fold for the two species although it too was increased 30-fold in the salinity treatment. Shoot growth was inhibited 52% and 17%, respectively. Doubling the [CaImedium at the time of salinization had no ameliorative effect on the growth inhibition and only attenuated the reduction in [Ca]l,,f (by 3.5% and 30%, respectively, in the two species). When H. vulgare was salinized at a low SAR (<28) and with equimolar Ca’+ and Mg” in the solution, the [CaIshoul decreased much more than the [MgIsh,,, (59 vs. 3 1%, Lazaroff and Pitman, 1966). Furthermore, the ratio of tissue concentrations in the shoot to those in the root decreased 74% for Ca”, but only 22% for Mg’*. In the four grass species, H. lanatus, L. perenne, D. glomerata and E rubra decreases in [Ca],,,, were 14,4, 0.24 and 3 times greater, respectively, than those for [Mg],,,, (Ashraf et al., 1990). In the two most salt-sensitive lines of Lophopyrum elongaturn the [Ca] of the flag leaves decreased 49% and 64% at high salinity, whereas the [Mg] had decreased 10% in one and increased 20% in the other (Omelian and Epstein, 1991). 3. Ca’ Translocation Effects: Correlation to Genotypic Sensitivity Translocation of Ca” to the shoot whether short term or net long term has often been more severely affected in salt-sensitive genotypes and cultivars than in relatively salt-tolerant ones. For example, in i? pratense the [CaIshootwas decreased more in the more salt-sensitive line (56% vs. 18%, Ashraf e f al., 1986). Of two F! vulgaris genotypes, the more sensitive had 22% greater [Ca]sh(,olthan the more tolerant at either of the two most moderate salinities (Lessani and Marschner, 1978). However, since shoot growth was reduced ten times more in the salt sensitive genotype and the [CaIFhoot was not reduced by salinization and nearly identical [ Ca],hoorin both genotypes at the highest salinity, it remains questionable whether Ca2+ was in short supply for the more sensitive genotype. In salt-stressed +

132

D.B. LAZOF and N. BERNSTEIN

B. vulgaris the [CaIshootwas reduced about the same in sensitive and tolerant genotypes after salinization for 19 days (NaCl stimulated growth taken as control, Marschner et al., 1981b). In Perseu americuna grafted on three differentially salt-sensitive rootstocks, [Ca],hooldecreases correlated to relative salt sensitivity (Downton, 1978). The situation is similar in monocots. In two Triticum species there was a large difference in the inhibition of 4sCa2t translocation to the shoot (90% vs. 53%, Davenport et al., 1997). This was correlated with the NaCl sensitivity of growth. Two genotypes of Z. mays were found not to differ in reductions of [Call,, (ca. 40%) after 3 weeks of salinization (Maas and Grieve, 1987). However, in two other genotypes [CaIshootof the more sensitive Z. mays genotype decreased nearly 50% after 18 days of salinization, whereas the [CaIrhootof the more tolerant genotype decreased only 30% (Cramer et al., 1994b). In field trials of H. vulgare the more salt sensitive of two cultivars had about a threefold greater reduction in [CaIlaf (mature leaf) after 3.5 months of growth under treatment conditions (Lynch and Lauchli, 1985). And in a solution culture study of the same two cultivars studied as week-old seedlings 5 h 45Ca2ttransport was reduced by 74% and 60%, in the more sensitive and tolerant genotypes, respectively (Lynch and Lauchli, 1985). In the three most sensitive lines of L. elongatum the [Ca] of the flagleaf at the intermediate level of salinity decreased 26%, 38% and 37%, but in the two most tolerant lines the reductions were only 13%, 35% and 9% (44%less reduction on average, Omelian and Epstein, 1991). In two Agropyrun species the [CaIshoo, decreased either 14% or 79% after 8 weeks of growth at 20 mM NaCl, for the more tolerant and sensitive genotypes, respectively (Elzam and Epstein, 1969). The [Ca],,,, of S. bicolor decreased more in the salt-tolerant than in the salt-sensitive genotypes (Grieve and Maas, 1988). At this early stage, the evidence is rather strong that uptake and translocation of Ca2+ is rather specifically affected by salinization, at least in the sense that this effect on Ca2+transport is usually stronger than the more thoroughly studied effect on K C transport, as well as both stronger and more consistent than any such effect on the transport of Mg2+. Similar to the growth inhibition, the effect on Ca2+ transport is usually much stronger on the nutrition of shoots specifically. Although the collected literature based on whole shoot analyses strongly suggests that a nutritional disturbance of Ca2+might have a role in the shoot growth inhibition and in the major mechanism of genotypic tolerance, assessment of metabolic disturbance within whole shoots will always be equivocal with regard to a growth effect (section LA).

IV. NUTRIENT TRANSPORT TO GROWING SHOOT TISSUE UNDER SALINITY 'Growing shoot tissues' means regions where either cell division, or cell extension are intensive. Within a meristem, mitosis dominates and cell extension is largely limited to that amount required for the maintenance of cell size (Baluska et al.,

THE NaCl INDUCED INHIBRION OF SHOOT GROWTH

133

1990, 1994). The SAM is responsible for primordia formation in both monocots and dicots, although in dicots it is more accessible to study. Growing leaves are usually heterogeneous systems containing both fully mature and extending cells. Particularly in monocots, the growing zone is often only a minute portion of a growing leaf, which includes discrete regions where cell division dominates, the leaf basal nieristem (LBM) and where cell extension dominates (the rapid expansion zone), rendering it a convenient system for investigating effects on particular developmental stages (section V1II.A). In much of the literature on Poaceae, effects on growth and metabolism are reported for whole ‘growing’ leaves, yielding little useful information. In dicots the leaves are also heterogeneous during most of the period that they are growing, but not in discrete macroscopically identifiable zones. In either dicots or monocots, but generally only for leaves which are either primordial or less than 1 cm in length can it be assumed that all cells are growing. Salinity-related data pertaining to shoot secondary and stem growth are extremely rare and are accorded little attention here. A.

PROTECTION OF GROWING TISSUES

Studies in growing leaf tissue, like those of whole shoots, have mostly examined the concentrations of only the salinity sources and K ’ , and only occasionally considered other nutrients (Table I). The idea that salt tolerance involves exclusion of Na’ and CI- is certainly more logical when applied specifically to growing tissues for at least there is a direct spatial association with the growth processes. However, other reservations about the ‘ion excess hypothesis’ remain problematic (section I.C.1). Most reports which have considered the exclusion of Na’ and CIfrom growing shoot tissues have raised the concept of protection, by which Na‘ and C1- are excluded from growing shoot tissues to a greater extent than they are excluded from the whole shoot (e.g. Jeschke, 1983, 1984; Curtis and Lauchli, 1985; Aslam et al., 1986). The concept of protection as a whole plant process has been given a solid basis in terms of physiological processes, especially in reports issuing from the laboratories of Jeschke and Pate over the last 10 years (see e.g. Jeschke and Pate, 1991a; Jeschke et al., 1992). Retranslocation, which plays a major role in this work, will be discussed specifically (section VI). As for whole shoot nutrition, as applied to young tissues the ‘ion excess’ hypothesis has often included the principle of disturbed K ‘ status (section I.C.l).Inclusion of other nutrients is more encouraging for investigations of young tissues, as more than one third included some nutrient other than K (Table I). Few studies have defined precisely the development of the young tissue analysed (Table I). Occasionally ‘young’ has included leaves which have neared full expansion. 8 . LEVELS OF Na AND K IN YOUNG TISSUE

Levels of Na do increase dramatically in growing shoot tissues on salinization. This is, however, not necessarily in conflict with the principle that these tissues may be protected. Growing tissues maintain levels of Na’ and C1- which are relatively

TABLE I Studies of disturbed mineral contents within young plant leaves as affected by salinization

Potassiumb Calcium'

Reference

Salt"

Aslam et al. (1986) Aswathappa and Bachelard (1986) Bernstein et al. (1995) Curtis and Liiuchli (1985) Delane el al. (1982) Durand and Lacan ( 1994) Fortmeier (1995) Greenway (1962) Greenway (1965) Greenway et al. (1965) Grieve and Maas (1988) Jeschke and Wolf (1985) Jeschke et al. (1992) Lazof and Lauchli (1991b) Lynch e f al. (1988) Maas and Grieve (1987) Martinez and Lauchli (1991) Martinez et al. (1996)

Yes Yes

Yes

Yes

Yes Yes Yes

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes -

-

-

Yes Yes

-

Yes Yes -

Yes -

-

-

Yes Yes Yes Yes Yes

Yes Yes Yes Yes

-

-

-

Other"

Def-Dev'

Comp-Mae' ConcRrans8

No No No No

Yes

Conc Conc

Yes Yes Yes No Yes No Yes No No No No No Yes No Yes No No No No No No No Yes No Yes Yes

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes Yes

Trans Conc Conc Conc Conc Conc Conc Conc Conc Conc Conc Conc Conc Conc Trans Trans

No No Yes No Yes No

Species A. amnicola Casuarina spp.

S. bicolor H. cannabinus H. vulgare G. max Z. mays H. vulgare H. vulgare H. vulgare S. bicolor Hordeum spp. L. albus L. sativa H. vulgare

z. mays

G. hirsutum L. sativa

Munns et al. (1988) Rawson ef al. (1988) Storey e f al. (1983) Treeby and Steveninck (1988) Wieneke and Lauchli ( I 980) Wilson et nl. (1970b) Wolf and Jeschke (1987) Wolf et al. (1 990) Ye0 and Flowers (1982) Ye0 e t a / . (1991)

Yes Yes Yes Yes Yes Yes Yes Yes Yes

Yes Yes Yes Yes Yes No No No No No Yes No No No No No No No Yes No

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Conc Conc Conc Conc Trans Conc Trans Trans Conc Conc

H. vulgare H. vulgare A. spongiosa Lupinus sp. G. max G. mmH. vulgare H. vulgare 0. sativa 0. sativa

Some studies were excluded for lack of any control treatment and others for SAR of all treatments. Otherwise the attempt was made to include all pertinent reports. Most studies were canied out following 5 days or more of salinization and many of the studies had a specific focus quite apart from disturbed nutrition of young leaves. Young leaves were considered to be any leaves which were still expanding at the time of specimen collection. "Whether levels of the salt elements were determined. b,'Whether [K], [Ca] were determined. dWhether the concentration of any other nutrient was determined and which. 'Whether the development of the young tissue was well defined by size (first yes or no) and by cellular development (second yes or no). Definition of cellular development for dicot leaves involves evaluation of whether they were in the pre-rapid expansion. rapid expansion, or slow phase of leaf expansion and for monocot leaves whether the leaf tissue was in the region of greatest elemental extension rates. 'Whether a comparison was provided of mineral concentrations in young tissue and mature (yes or no). YWhetheronly nutrient concentrations were determined. or whether transport of nutrients was provided (conc or trans). Transport could be either net gain over a defined time, or short-term transport of mineral tracer.

136

D.B. LAZOF and N. BERNSTEIN

lower than those in mature tissues and still experience drastic increases in Na' and C1- levels. The data are more conflicting, however, regarding whether there is a corresponding decrease in [K] in growing shoot tissues, such as that which occurs in the more mature shoot tissues. In salt-stressed H. cannubinus compared to '0 Na' control plants, [K],,af showed about a 5% increase in the youngest leaves (Curtis and Lauchli, 1985). In salt-stressed A. amnicola the Na:K ratio increased fourfold in the youngest leaves after 3 weeks of stepwise salinization, however 85% of the Nat was contained in the bladders on the surface of the laminae (Aslam et al., 1986). In L. ufbus,the youngest leaflets had about the same percentage decrease of [K] as mature leaflets in plants salinized for 7 weeks, as compared to 5 0 p M Na-grown control plants, although Na content of the youngest leaflets was slight in comparison to mature leaves (Jeschke er al., 1992). The Na content was 80 pmol leaf-' in those leaflets which were expanding at more than twice the rate of the next youngest leaves (Layzell et ai., 1981; Jeschke er af., 1992). Of particular interest are the reports which have examined Na and K levels of young tissues at both a growth-stimulating and growth-inhibiting level of salinization. In Arripfex spongiosa "a] of young leaves increased slightly (30%-40%) as plants were moved from growth stimulating to growth inhibiting salinity levels for 7 days (Storey et al., 1983). The [K],, did not seem to decrease. Levels of and [KlIed in two size classes of minute leaves (either < 0.5 mm or 1-1.5 mm in length) of L. sariva 2 and 5 days after commencement of salinization from the control level of 10 to the 80 mM NaCl level were studied by electron probe x-ray microanalysts (EPXMA) (Lazof, 1991b). Five days after initial salinization "a],& was consistently higher than it was in the 10 mM NaCl control plants (on average nearly fourfold and up to 17 pmol (g fresh weight)-'). Changes in the "a],,, after just 3 days of commencing salinization were less consistent. The [K],,,, tended, however, to be reduced in both classes of leaves and at both sampling dates (on average decreasing 16%). The first trifoliate of G. max, salinized to 66 mM NaCI, showed >12-fold increase in 30 h accumulation of 22Na+over the same leaves grown at 7.5 mM NaCl (Wieneke and Lauchli, 1980). Similarly, in the expanding second trifoliates of G. max [Na],,,, increased half as much as in the first trifoliates after salinization to 25 mM NaCl (Durand and Lacan, 1994). In Clycine wightii and Glycine tomentellu the [Nallearin young leaves rose > 20-fold after 4 days of salinization to 80mM NaCl over the control levels of plants grown with 0.5 mM NaCI, whereas the [K],,, in these species increased during the same period in these leaves (Wilson et al., 1970b). In Poaceae, the situation is much the same as that described for dicots. Greenway (1962) reported that the young shoot tissue (leaf numbers > 3, sheaths and the stem apex) had large increases in [Na] after 5-15 days of salinization (5-15-fold over the levels in 1 mM NaC1-grown control plants). These young shoot tissues responded with about an 80% reduction in Na content and a 40% increase in K content when, after 5 days of salinization, plants were set into a '0 Na' solution for 5 or 10 days. These alterations were associated With 19-31% increased net growth of the same plant parts during the 5 or 10 day growth periods. Only a small portion of the

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

137

decreases in levels of Na and C1 could, therefore, be accounted for by growth dilution (i.e. decreased concentration due to relatively greater volume expansion). The combined young shoot sample weighed only 30mg dry at the time the plant was transferred to '0 Na' solution. These data support the idea that ion relations of the young shoot tissue are well correlated with growth effects (3 day time frame). After a longer period of salinization young leaves of Hordeutn vulgare which had emerged 10 days after salinization had a leveling off of [K] at about 25% of the value of [K],,, in the same leaves of the '0 Na' control plants (Jeschke and Wolf, 1985). Compared to a 1 mM NaCl control, the [KIleatin young leaves of H . vulgare had been reduced about 50% by salinization to 125 mM NaCl; however, the reduction in [K],,,, was 20% for the youngest group of leaves when sampled within two weeks of emergence (i.e. two weeks since emergence for the oldest leaves in the 'young' group, Greenway et al., 1965). Fourfold increases of [Nailed+ in the 2cm basal leaf zone of barley could be measured whether or not the salinity treatment had altered growth (4 and 6 day treatments at a moderate 80 mM NaCl (Lynch et nl., 1988)). Neither the differences in [KIle.,, nor the other two nutrients which were measured were significantly affected by salinization, although the experimental errors were large. Within the rapidly elongating region of a barley leaf the levels of Na' and C1- could be substantially reduced (ca. 50%) within 2.5 h of initiating a stepwise reduction of the salinity in the nutrient solution (Munns er al., 1982). The [K], however, did not change, although a large increase in leaf elongation rate could be measured during the same time frame. In other Poaceae, results have been similar. Bernstein et a / . (1995) reported a 20% decrease in the deposition rate of K' and a substantial Na+ deposition rate (ca. 1.6 nmol mm-' hK') in the youngest tissue (4mm from leaf base) of expanding leaves of S. bicolnr. This represented a greater than 15-fold increase over the rate in the same tissue of the 1 mM NaCl control plants. Although growth rates during the first hour have not always been found to be indicative of enduring growth inhibitions (sections I.B.2, II.A), it is remarkable that the ion content of the elongation zone responded so quickly (2.5 h) to a decrease in the salinity of the rooting medium. Earlier, with a less precise definition of 'young tissue', five weeks of salinization to 86 mM NaCl led to considerable [Na] in the young sorghum leaf tissue (20-80 pmol (gFW)-', Grieve and Maas, 1988). In 0. sariva accumulation of "Na' following a 48 h labelling through the root medium was also much lower (per gram tissue) in the youngest leaves after 8 days of salinization (Ye0 and Flowers, 1982). In Triticum aestivum the net K t deposition rates into 5-10mm zones of a young leaf were not affected by a very moderate salinization (effect on growth not reported, Hu and Schmidhalter, 1997). C. DISTURBED Ca STATUS IN YOUNG TISSUES

In G. m u 4sCa2' transport to the young first trifoliate leaves was reduced 67% by salinization to 66 mM NaCl, compared with a 7.5 mM NaCl control (Wieneke and Lauchli, 1980). In the youngest leaves of Lupinus albus grown for 23-33 days with

138

D.B. LAZOF and N. BERNSTEIN

40 mM NaCl leaf, Ca content decreased 62% (control of 0.5 mM NaC1) and only about 30% in older leaves (Jeschke et al., 1992). The decrease in Ca content was greater than that for either K or Mg (SARC56). Decreases of [KI1,,, were only 45%. In minute leaves of L. sativa, [Ca],,,, was determined 2 and 5 days after commencement of salinization from the 10 mM NaCl control level to 80 mM NaCl (Lazof and Lauchli, 1991b). In leaves of 1-1.5 mm in length [CaIlrafdecreased 62% and 33% when measured after 3 or 5 days of salinization, as determined by EPXMA of freeze-dried apices. In young leaves of H. cannabinus trends tended towards decreasing [Ca] after 6 weeks of salinization at 75 m M NaCl (Curtis and Lauchli, 1985). In field grown, salt-sensitive H. vulgare [Ca] of the youngest shoot tissue (sheath 5, 90 days after sowing) decreased 50% after a twofold increase in [Na],,,, (Lynch and Lauchli, 1985). In a more tolerant genotype there was no decrease from the control [Ca],, at any stage of development. The elongating region (2 cm leaf base) of barley leaves tended towards decreasing without significant effects on ion contents in the moderate salinity treatments (up to 80 mM NaCl, Lynch et al., 1988). The lack of significant effects may have been partially due to the relatively large 2cm segments used (section 1V.E). In Z. mays the [Ca] in ‘immature’ leaf blades decreased by 75-90% whereas the [K] was unaffected (Maas and Grieve, 1987). Elsewhere the [Ca] of the youngest leaves decreased 78% and 68% after 15-19 days salinization by 100mM Na’ (added as the CI- or SO,’salt, respectively, Fortmeier, 1995). In salt-stressed S. bicolor, Ca2+ deposition decreased 94% as [Ca] decreased 75% in the youngest region (3-9 mm from the base) of a leaf which was a primordium at salinization (SAR = 63, Bernstein ef al., 1993b, 1995). The [K] of this tissue decreased only 20% as much as [Ca] in the first few millimetres. In ‘immature leaves’ of a relatively salt-sensitive S. bicolor genotype [Ca] declined 9&95% whereas [KIleafdecreased merely 10-12% after 5 weeks at 86 mM NaCl (Grieve and Maas, 1988). It was recently reported that in ‘I: aestivum salinization actually increased the net Ca2’ deposition into discrete zones of the leaf; however, the very moderate salinization included a high [Calmed,um and a growth effect was uncertain (Hu and Schmidhalter, 1997). Relative elemental growth in young leaves could be preserved in 3. bicolor (at control level) during salinization by supplementation of [CaImedlum (changing SAR from 63 to 37, Bernstein el al., 1993a). Additional work also showed that the profile of [Ca] in the elongation zone was restored by supplementary [Ca]medlull, (Fig. 2)

D. OTHER NUTRIENT DISRUITIONS IN YOUNG SHOOT TISSUES

Apart from Na, C1, K and Ca, data on salinity effects on the nutritional status of young tissues are largely limited to Mg and P (Table I). In young leaves of L. albus Mg content decreased about 50% after salinization for several weeks (about the same reduction as K content, Jeschke et al., 1992). The reduction for Mg was not more severe in young leaflets and may largely have been associated with reduced

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH I

'

I

'

0

'

I

control

supplemental Ca

0

m 2 c

I

139

a .

40

0

10

20

*

-=---~

30

40

50

Distance from leaf base (mm)

Fig. 2. Calcium distribution profile in young leaf of S. bicolnr 4 days after salinization to 100 mM NaC1 with or without an additional 10 mM [Ca],r,r.d,u,r,.

growth. The Mg content of a leaf which had just finished expanding at the time of harvest in A. umnicvla had decreased 40% relative to optimally salinized controls (Aslam er al., 1986). In S. bicolor Mg deposition in the youngest leaf regions was reduced 74%: (Bernstein ef d., 1995). This was 22% less reduction than the decrease in Ca deposition, but almost fourfold that for K. In Z. mays the [Mg] of young leaves showed no consistent relationship to salinity level after a 5 week treatment (Grieve and Maas, 1988). The [PI of young leaves (emerging during salinization) was reduced 23% in a salt-sensitive Glyciize species after 4 days exposure to 80 mM NaCl, whereas [N] was reduced just 13% and [K] not at all (Wilson et ul., 1970b). In young L. lureus leaflets, [Plleafincreased 125 and 34% 23 and 30 days after salinization to 50 mM NaCl, although had decreased 17% after 16 days ('0 Na' controls, Treeby and Steveninck, 1988). In L. albus there was no consistent alteration in P content of young leaves due to salinization (Jeschke et a/., 1992). Three hour transport of 31P to small leaves ( 130 mg fresh weight) of Gossyhrn hirsuturn decreased 24% after an 8 day salinization (SAR of 62), although a much larger reduction occurred at extreme salinity (SAR > 200, Na:Ca = 150. Martinez and Lauchli, 1991). In Lactuccr sativu transport of .'>P to young leaves was inhibited after moderate salinization compared to IOmM NaCl (SAR of 41, Martinez er al., 1996). In Z. ntuys [PI increased 68% in immature blades aftcr 3 weeks of salinization to 86 mM NaCl (Maas and Grieve, 1987). Net P accumulation in discrete zones of a young iT: nestivum leaf was not affected by a moderate salinization (growth effect also uncertain, Hu and Schmidhalter, 1997).

E. EFFECTS IN YOUNG TISSUES COMPARED TO EFFECTS IN MATURE TISSUES

A number of studies have compared disturbed nutrition of young shoot tissues (where it is more reasonable to search for immediate causes of a growth inhibition) with more mature tissues or whole shoots of the sample plants (Table I). For leaves

I40

D.B. LAZOF and N . BERNSTEIN

emerging during salinity stress in two Glycine species, increases in [Na],,,, and [Cl],,, were about half and the decrease in [K],,,, about one third that in already emerged leaves after a moderate salinization (Wilson et al., 1970b). In H. cannabinus decreases in [Calledtended to be less in young leaves than in mature leaves, whereas [NaJleafincreased nearly sevenfold and reductions in [KlIed were nearly 70% less in the least developed leaves (Curtis and Lauchli, 1985). In salt-stressed A. amnicola the K/Na ratio for rapidly expanding leaves was one-third that of mature leaves (Aslam et al., 1986). In A. spongiosa there was no apparent change in [K],,, or [P]l,,t of the very young leaves, but a large decrease in [KIleAr and [PIleJfof the mature leaf tissue (Storey et al., 1983). In L. albus the leaf content of Ca, unlike either that of K or Mg, decreased twice as much in the youngest group of leaves, although P content decreased in the young leaves, but increased in stems and petioles of mature leaves (Jeschke er ul., 1992). In Lactucu sutiva transport of 32P to young leaves was inhibited to approximately the same extent over a range of leaf developmental stages with moderate salinization (Martinez et al., 1996). however 3 h transport of 32Pto small leaves of G. hirsuturn was decreased only half as much in the youngest leaves as it did for the whole shoot (Martinez and Lauchli, 1991). The youngest region of barley leaves (0-4 cm), maintained similar [Nailed and [C1]l,,f but had 35-50% higher [K],,,, than tissue regions more than 4 cm from the base (Delane et al., 1982). Elsewhere net K' transport into the two youngest barley leaves was reduced somewhat more than in older leaves (Wolf and Jeschke, 1987). In salt-stressed S. bicolor, Naf deposition was highest 8-18 mm from the leaf base in a leaf which had been a primordium when salinization began (Bernstein et al., 1995), corresponding approximately to the region of maximal elemental growth (Fig. 7b, day 1, in Bernstein et al., 1993b). The reductions in Ca2+deposition were largest for segments 3-6 mm from the leaf base (Bernstein et al., 1995). Previously [Call,,, had decreased 2.5-fold more in young than in mature S. bicolor leaf tissue (Grieve and Maas, 1988). In Z. mays the [Na] in immature leaf blades was only 56-72% as great as in mature leaf blades, whereas reductions in [KI were 10-37% greater in the mature tissue (Maasand Grieve, 1987). The [PI of the young blades increased 22% less than in more mature tissue. Elsewhere, [Ca] decreased two to three times more in young Z. mays tissue than in the whole shoot, whereas [Na] of the youngest leaf tissue averaged 30% less after 4 weeks of salinization (Fortmeier, 1995). At first sight these results appear quite mixed, with nearly as many cases of greater change as lesser change for nutrient or ion status in young growing tissues compared with older shoot tissues. However, the results consistently indicate less accumulation of Na' and C1- in younger tissues, as well as less decrease in [K]. It should also be kept in mind that changes in net ion accumulation are as much a product of reduced biomass production, as of alterations in transport. Furthermore, changes in P transport due to salinity are heavily dependent on the solution [PI and often increase due to salinization especially in older tissues (section V1I.D). With these points in mind the data consistently demonstrate that for a variety of plant species (dicots and monocots) transport of Ca2' to young tissue is inhibited by

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

141

salinization, although a growth-related nutritional disturbance is likely to be masked whenever whole shoots or whole graminaceous leaves are bulk analysed. The dependency of detecting a nutritional disturbance on consideration of tissue development also suggests that within still smaller and more precisely defined growth zones the divergence from whole shoot effects might be much greater still. As shown in Table I most of the studies which have included salinity effects on nutrient status or nutrient transport to growing tissues, have not precisely defined the development of the 'young' tissue. F. GENOTYPIC SALINITY EFFECTS IN YOUNG TISSUES

The more susceptible of two G. mrw cultivars transported two to three times as much "Na' to the youngest expanding leaves during 12 or 30 h, whereas 30 h 45Ca2i transport was also more inhibited (67% vs. 60% inhibition at 66 vs. 7.5 mM NaCl) for this genotype (Wieneke and Lluchli, 1980). In young leaves of two Giycine species, [PI was differentially affected, being reduced by 23% and 10% in the more sensitive and tolerant species, respectively, after 4 days of salinization to 80 mM NaCl (Wilson et al., 1970b). There was no apparent difference between the two species in Na and C1 levels of the young leaves in the more moderate treatment. In H. cannabinus the [Ca] of the younger leaves tended to decrease less in the more salt-tolerant genotype (3% vs. 9% reduction), with only minor differences in growth inhibition (Curtis and Lauchli, 1985). In monocots, the evidence for NaC1-induced nutrient disruption in growing tissue seems weaker. There was a decrease of about 60-70% in the salinized [K],,, compared to the '0 Na' controls either in late or early developed leaves for either of two differentially sensitive H. vulgure genotypes (Jeschke and Wolf, 1985). In two other genotypes of H. vulgare there were no effects on ion accumulation in an expanding leaf zone, although there was also no differential growth effect, disregarding the most extreme treatment (Lynch et al., 1988). In two genotypes of S. bicolor the [Ca] of the younger leaf tissue actually decreased 25% more in the more salt-tolerant genotype after 5 weeks of salinization to 86 mM NaCl (SAR of 62, Grieve and Maas, 1988). In a study examining ion status of young leaves in Z. mays genotypes, the [Ca] decreased by much more (23% and 6%) in the youngest leaves of the more salt-sensitive Z. mays genotype with salinization to 100mM NaCl or 50mM Na2S04, respectively (Fortmeier, 1995). Elsewhere the [PI of immature leaf blades increased much more for the more salt-sensitive genotype (100% vs. 37%, Maas and Grieve, 1987). G. LACTUCA SATIVA: A MODEL DICOT SYSTEM

Changes in growth, ion content and net ion transport to particular leaves of varying developmental stages of L. suriva were studied during the first few days of a moderate salinity stress (80 mM NaCl treatment with SAR of 56) (Fig. 3, Lazof et al., 1991). This species' relatively small size and rapid leaf emergence and

142

D. B. LAZOF and N. BERNSTEIN

I

7

13

14

Leaf number (younger +z-

1

8

9

10

11

12

Fig. 3. Concentrations of K, Ca and Mg in leaves numbered 7 to 14 of salinized L. on either 15 or 18 days after transferring seedlings to solution culture. Salinization with 80 m M NaCl commenced stepwise on 13 DAT and ended on 15 DAT. Concentrations are expressed as percentage of the ion concentrations in the same leaves of control plants growing with 10 mM NaCI. The coefficient of variation averaged 0.17 for all data shown ( n = 3 or 4 for individual leaf numbers of each treatment).

sativu

expansion rates allow convenient study of the response of mid-vegetative plants. Response of ion status to salinization has been measured along with nondestructive continuous analysis of individual leaf growth in the 'loose leaf cultivar Black-seeded Simpson in which access to whole leaves and negligible stem mass allows whole shoot growth to be calculated as well (Lazof et al., 1991). The fact that a large number of leaves grow simultaneously, allows evaluation of stress effects on leaves which were exposed to salinity at a range of leaf developmental stages. Salinity stressed plants were exposed stepwise to 80mM NaCl and compared to 10 m M NaCl 'control' (growth stimulated) plants. The salinization began 13 days after transferring (DAT) seedlings to solution culture and was completed 30 h later. In Table 11 growth data are summarized indicating the time at which individual leaves passed out of a rapid exponential expansion phase and the rate of expansion. These rates are relative leaf expansion rates based on fresh weights which were calculated from mid rib length according to empirical equations (Lazof et al., 1991). Subsequent to the exponential expansion, relative leaves continued expanding over the next several days at rates just 30% of the rapid rates (Lazof, unpublished). The deviations of leaf ion concentrations from those in control were determined

I43

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

TABLE I1 Solinization effects on the leaf growth of’Lactuca sativa Salinity

Leaf numbers

10 m M NaCl

6-8

80 m M NaCl

9-1 I 12-14 6-8 9-1 1 12-14

RGR (day

.I)“

1.015 ? 0.03 1.104 ? 0.02 1.155 2 0.05

0.973 ? 0.007 0.982 2 0.019 0.996 t 0.07

Day of exit’ 13-16 16-18 18-2 I 14-16

17-18 19-2 I

“Relative growth rates for groups of leaves during the phase of constant exponential leaf expansion (Lazofet NI.. 1991). Errors are standard errors of mean ( n = 12). ”The day at which the leaves exited from the constant exponential phase of expansion. range is from oldest to the youngest leaf within each leaf group.

for three nutrients in individual leaves of salinized plants (Fig. 3). Leaves were destructively harvested at either 15 or 18 DAT (18 or 90 h after the completion of salinization) and ion contents analysed by inductively coupled plasmometry. Data are shown for Kt,Mg” and Ca”. Ion concentrations were noticeably more depressed for Ca” than for either K’ or for Mg”. Net deposition, which is composed of both growth and ion transport effects, was affected by salinization (Fig. 4). The primacy of the salt stress inhibition on nutrient transport compared to the effect of organ growth (sink strength) was suggested by comparison of the deposition of these three cationic nutrients. By far, the net Ca2+ transport was the most severely affected by the salinization treatment. Reductions in net Ca” transport during the 15-18 DAT period averaged 13 and 41% more than the reductions in net K ’ and Mg2” transport, respectively, for the six rapidly expanding leaves shown. Reductions in net CaZt transport were, on average, 22 and 70% greater than the corresponding reductions of K + and Mg” transport. Furthermore, of K’, Ca2‘ and Mg”. only Ca” transport seemed to be more severely reduced (10% more on average) in the youngest leaves. The developmental stages of these leaves are shown in Table 11. Leaves 13 and 14 had fresh weights of less than 50 mg at I8 DAT (even in the control plants). More minute, recently initiated leaves were analysed by EPXMA (section IV.C, Lazof and Lauchli, 199I b). Complementary data from these studies for P and S are given below (section VI1.D).

H. SUMMARY: SALINIZED NUTRITION OF GROWING SHOOT TISSUES

As in whole shoot studies, much of the focus, in the study of ion relations in young tissues has been on Na+ and Cl-, with half considering either Kt alone or no nutrients at all (Table I). Often, also, due to comparison of salinized and ‘0 Na’ treatments, alterations in mineral nutrients associated with growth inhibition could not be properly assessed (section 1II.A.I). The study by Wieneke and Lauchli

144

D. B. LAZOF and N. BERNSTEIN

16

60

12

40

8

20

4

7

8

9

12

13

14

13

14

I

6

52

Calcium

7

k-

12

3 8 04

E

4

02

C

.-E

Magnesium

l

7

8

I

9

12

13

14

Leaf number Fig. 4. Net nutrient deposition rates of six leaves of L. sativa from 15 to 18 DAT in salinized plants. Leaves 12 to 14 gained 99% of their mass during this period and were assumed to have gained 90% of their final nutrient composition during the same period, to conservatively estimate net transport. The average coefficient of variation for leaf fresh weight was less than 0.2. The errors associated with ion concentrations are shown in Fig. 3 (n = 4, Lazof et al., 1991).

(1980) might be taken as an early model in terms of experimental design for testing alterations in ion transport to young growing tissues within the 3 h to 3 day time frame. Several-fold increases in Na transport to young soybean leaves were documented after just 30h of salinization to 66mM NaCl, as compared with a 7.5 mM control. The principle of rising levels of [Nal in young leaf tissue within a few days of moderate salinization, has also been indicated in Lupinus, Lnrctuca, Hibiscus, Atriplex, Hordeum, Sorghum, Oryza and Zea. Although, even in young leaves, one might expect [K] to decline with the availability of a less expensive osmoticant (section I.C.2), there have been nearly as many instances of increased

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

I45

[K] of young tissues (section 1V.B). Several of the studies have been equivocal, leaving open the question of whether techniques were sufficiently precise to measure potential differences. Probably because of the nature of net transport studies, few of the studies considering changes in [K],,,, were resolved to the first few days of salinization. Four studies did study salinity effects on K+ transport during the first week (Greenway, 1962; Wilson et al., 1970b; Lazof and Lauchli, 1991b; Bernstein et al., 1995) and all of these except the latter reported a 1&20% decrease in [K] within that time frame. Although there are many doubts about the significance of changes in [Na] and [Cl] and the increase in [K] subsequent to salinization (sections I.C. I , I.C.2). these studies do establish a baseline for possible broader effects on nutrient transport and status. The evidence may be stronger for effects on nutrients other than K t . A 30 h effect of salinization on Ca” transport (67% reduction) was demonstrated in soybean (Wieneke and Lauchli, 1980). Similar results (drastically reduced Ca2 ’ transport and status) have been demonstrated in Lupinus, Lactuca, Hibiscus, Hordeum, Sorghum and Zea, although often in a longer time frame (section 1V.C). Some disruptions in supply of other nutrients have also been reported. Five studies were discussed wherein Mg’+ transport or Mg levels were reduced by salinization with reductions ranging from 22 to 74% (section 1V.D). With regard to P status and transport to young shoot tissues there have been four cases of inhibition (17-23%) and four cases of augmentation (30-125%). The data for the effects on P nutrition seem to be strongly affected by experimental details such as duration of salinization, exact developmental stage of the ‘young’ tissue, severity of salinization and solution levels of Ca’ ‘ and Pi (section V1I.D). The current evidence that the NaC1-induced inhibition of shoot growth in any plant species is due either mainly, or largely, to disturbed nutrition, is not compelling. Rather, the evidence to date is scant, often indirect, often lacking proper controls and often limited by a number of experimental design features. However, at this early stage of investigation there is quite a lot of evidence to suggest that disturbed nutrition may be a major factor in the limitation of shoot growth and that these effects are not limited to those ions which could be expected, a priori, to be altered under salinity stress (Na’, CI-, K’ and, possibly, Ca2+ in the case of studies at extreme SARs). A few studies have tested the idea that nutrient disruptions in young tissues might be controlled or avoided in more salt-tolerant genotypes (Wilson et al., 1970b; Wieneke and LLuchli, 1980; Curtis and Lauchli, 1985; Maas and Grieve, 1987; Lynch et al., 1988; Fortmeier, 1995). The question of salt-tolerant genotype is crucial and needs much more careful consideration in the coming years. At this early stage of investigation, the cultivar differences have been small, at best, and often statistically insignificant. Quite possibly, this lack of evidence may be entirely due to a lack of appropriately sensitive methods being applied to the question, as well as to inadequate interest and lack of resources having been allocated to the most promising techniques. The chief limitation associated with the data currently available and relevant to salinization effects on growth-related mineral nutrition, is that there are so few

146

D. B. LAZOF and N. BERNSTEIN

studies (Table I). Less than half have considered a nutrient other than K’. Very few short-term studies have been conducted using tracer techniques, so that little is known about the early onset and time course of nutritional disturbances. Among the few studies which have employed tracers for considering effects of salinization on transport, only one has considered effects of salinization within a time frame of less than 3 days (Wieneke and Lauchli, 1980). Less than 15% of the studies have precisely defined the developmental stage of the growing tissue during the treatment, even though nearly half provided some indication of the organ’s development in terms of size (Table I). The use of ‘young leaf has included everything from primordia to leaves completing expansion. Rapidly expanding tissues in the shoot are also rapidly undergoing vacuolization and it may be necessary to consider compartmentalization of nutrients within these tissues before meaningful interpretation about nutrient disruptions, or modifications from the control levels in the most relevant metabolic cell phases, the cytoplasm, in extending cells. An even more uncharted territory is that of the nutrient status of and nutrient transport to the unvacuolated and rapidly dividing cells of shoot meristems. Previous reviews have also discussed the effects of salinity on ion accumulation in growing leaves even though the topic was not central (Munns and Termaat, 1986; Munns, 1993). Because of the difference in focus and the consequent briefer attention to experimental diversities, those reviews reached divergent conclusions regarding NaC1-induced effects on mineral nutrition. It would not be useful. however, to delve once more into the importance of such design details as localization of effects, time frame, appropriate controls, nutrients selected, SAR etc. (sections LA, 1.C. ILA, 1Il.A) in specific relation to these excellent reviews, or the reports cited therein. Readers should consider the earlier arguments in light of the details we have been able to include here.

V. THE SHOOT MERISTEMS: SPECIAL NUTRIENT TRANSPORT CHALLENGES The transport challenges occurring in the SAM and in the LBM of monocots are much the same, since in both cases the meristems are encased in layers of developing tissues and subject to only very limited transpiration. In some dicots inner leaves even up to a few millimetres in length do not transpire, as they are wrapped by several layers of larger leaves, as well as leaf trichomes directly over the apex (Wiebe et al., 1977; Collier and Huntington, 1983; Lazof and Lauchli, 1991b). In monocots, the leaf‘s growing zone is enclosed inside a whorl of older leaf sheaths and does not transpire. Following emergence from the whorl of older leaf sheaths, the transpirational area of the whole young leaf increases steadily. Since the apex (meristem plus primordia) of both monocots and dicots is practically non- transpiring, the identification of the driving force for nutrient transport itself

THE NitCI INDUCED INHIBITION OF SHOOT GROWTH

147

still requires additional study. Cell expansion itself may possibly lower water potential and drive water flux (Nonami and Boyer, 1989; Matyssek and Maruyama, 1991; Nonami et d., 1997). The primary walls within the apical 50 p m of a dicot SAM are very thin (on the order of 0.1 mm, Lazof and Lauchli, 1991b) and of similar dimension close to the monocot leaf base. This implies that apoplastic flow must be more restricted in these tissues than through much of the shoot (Esau, 1977; Bernstein ef d., 1995). At the same time the nearest functional vascular elements can be 800 p m or more from the meristem (Lazof and Lauchli, 1991b). Nutrient transport must either occur, then, through the thin primary cell walls or symplastically. This would present an especially serious challenge for those nutrients which are thought to be virtually non-tansportable through the symplasm (e.g. Ca' ' , Hanson, 1984; Clarkson. 1993). Although the monocots have developed vascular elements traversing the expanding zone, cross-linking lateral veins may not be functional within the expanding zone (Barlow, 1986) and the longitudinal veins may need to supply an area much larger than within mature leaf zones (Bernstein et a/., 1995). In and very near the meristem, cells are not vacuolated. There is vascular discontinuity between the stem and the leaf vessels in the cell division zone of the leaf base, requiring that transport through the leaf division zone and into the developing vascular systems of the expanding zone rely either on symplastic transport or transport through thin cell walls. The requirement for continuous nutrient supply to maintain the mineral status within rapidly expanding tissue renders the meristematic region highly susceptible to nutrient disturbances. This necessitates that the levels being maintained are entirely cytoplasmic and that they exist without any local mineral reserve. Supply, then, must closely match the needs of the process of cell division and the mineral composition of each daughter cell. This compartmentational aspect of meristematic tissues has a certain advantage for the physiologist in that the whole meristematic volume is largely cytoplasmic. however microtechniques may still be required, both because the meristematic volume itself is minute and because subcellular volumes such as the nuclear phase become relatively major. Beyond the challenges to meristematic nutrient transport which occur under near optimal conditions, additional limitations may pertain during salinization. For example, salinity was found to reduce the width of the conducting elements in the expansion zone of sorghum leaves. Protoxylem cells in the central vascular bundle had a radius of 12.6 p m in the control leaves. but only 9.1 p m in salinized leaves 1.3 cm from the leaf basc (N. Bernstein, personal communication). In this same location, studies with fluorescent dye demonstrated that whereas 96% of the vascular bundles were functional in transporting water in control plants, only 56% of the bundles were actively conducting in the salinized plants. Salinity had no affect, however, on the number of vascular bundles across the leaf growing zone and actually advanced development of the transport anatomy. The concept that salinity may have specific effects on phloem transport, or nutrient recirculation is explored below.

148

D. B. LAZOF and N. BERNSTEIN

A. THE NUTRITION OF RAPIDLY DIVIDING CELLS: POSSIBLE EFFECTS OF SALlNlTY

There are few published studies of nutrient status of shoot meristems and still fewer which have considered the effect of salinization within these minute tissue volumes. In the study of salinization effects on the nutrient status of shoot meristem, Storey et (11. (1983) used EPXMA to examine meristematic leaf tissue in Atriplex spongiosn, a halophyte bearing salt bladders. There was little effect on [PIl,,, or [S],,,, when roots were exposed to 600mM NaCl for one week or 400mM for two weeks and compared to leaves from plants cultured at growth-stimulating NaCl levels. The stability of the nutrient levels in the meristematic tissue of this halophyte, occurred despite a 60% increase in the Na:K ratio in the whole shoot as salinization was increased from 200 to 400 mM NaCl. This investigation analysed ion levels in frozen hydrated cryofractured specimens, a technique which can still be considered state of the art for botanical EPXMA. The relative levels of P and S could be deduced from the ion ratios which were determined from the peak height ratios in energy dispersive x-ray spectroscopy (EDS). No effort was made to consider [Ca],,,, or [Mgll,,,, nor was there any attempt to estimate concentrations by comparison with spectra from frozen standards. The study was not focused on effects within the meristem. Although the report did feature some secondary electron images (SEIs) of mature leaf tissue, none were shown for the meristematic tissues. Meristematic cells were identified by the absence of vacuoles. Such cells reportedly occurred among cells having either a few small vacuoles or larger central vacuoles. In a subsequent investigation using EPXMA of frozen hydrated cryofractured specimens from salt-sensitive lettuce, Lazof and Lauchli ( I991 b) demonstrated that after just two days of salinization there were significant decreases in [K] throughout the SAM, as compared to the control plants grown with I O m M NaCI. The decreases in [K] were close to 35% throughout the apical 100 pm. Even in the most apical 10 pm, [K] appeared to have decreased about 30% (ca. 16 pmol (gFW-') during the two day salinization, while [Na] increased about threefold (ca. 15 pmol (gFW)-l). Decreases in [PI of 20% were also indicated in the most apical 10 p m and up to 37% in the underlying 50-200pm below the absolute apex. These alterations from control levels were much the same after an additional three days of salinization, indicating that nutrient supply was not increased by plant adjustment to the saline conditions during the first week. The morphology and anatomy of the SAM as analysed in SEIs of frozen hydrated cryofractured specimens clearly showed that the most apical 40 p m were populated exclusively by non-vacuolated cells and that there were no central vacuoles within the first 150 pm. The most apical 10 p m was composed of a thin cuticle (ca. I pm) and cells with diameters ranging between 5 and 12pm, the smaller cells being largely nuclear. This microanalytical study, unlike the earlier study in Arriplex, was focused on the ion relations of the meristem. The spectra acquired from the apices were compared with spectra from frozen standards in order to estimate ion levels and an automatic algorithm for determination of peak location, peak width and background levels had been developed (Lazof, 1991a).

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

149

Due to an interest in determinations of additional nutrients with smaller EDS peak heights and in analysis of relatively large structures such as just initiated leaves, complementary EPXMA was carried out in freeze-dried specimens (Lazof, 1991a,b). In the most apical 200 pin the [Ca] showed a tendency to decrease (up to 38% after several days). However, 500 pm below the apex, where the isolation of cells from vascular elements would first arise, the [Ca] had decreased 69% in the salinized plants after two days of salinization. This reduction was maintained during three additional days of salt stress. The severity of the reduction in [Ca] at this distance from the meristem was similar to that detected in leaves measuring between 1 and 1.5 mm in length. Possibly, in the most apical 200 pm a restricted supply of Ca2+ might quickly inhibit cell division and expansion of that tissue, so that levels do not decrease further due to expansion. More fluctuation of the [Ca] would be possible at a greater distance (500 pm) where the cells contain central vacuoles. The data suggested that a decrease in [Mg] of 80% at locations 200 pm below the apex during the first two days of salinization may be largely (70%) overcome during an additional three days of salinization. The effects of salinization on the nutrient status in shoot meristem cells has also been studied by bulk chemical analysis of minute samples. Although few investigators have shown interest in this approach, it is possible to include short-term (radioisotope) transport in such studies. This constitutes one very important advantage over EPXMA which does not allow any isotopic analysis and in which sensitivity for analogues is rather low (Lazof et al., 1992). In the dissected SAM of lettuce grown under similar conditions as in the EPXMA study, there was also a tendency towards reduced short-term "P transport after salinization for 5 days (Martinez et a/., 1996). The inhibition of 3 h 32Paccumulation in the meristem was apparently less than the inhibition in whole plant uptake of '?P due to salinization. For the whole shoot, transport was reduced 28%, but only 13% on average within several loosely defined 'meristem segments'. Short-term P, transport was reduced 1845% when plants were transferred to an unlabelled solution for 3 h after the 3 h exposure to 32P.The greatest reductions were in the 'outer part of the meristem base', suggesting that salinization may have affected the ability of the underlying vascular elements to supply P, to the rapidly dividing cells. Effects on Pi transport from salinization for time periods less than 5 days were not studied. The five apical segments which were separately analysed were not well defined in terms of dimension, location or cellular development. In the same species and genotype it was earlier shown that analysis of cells devoid of vacuolization would have required excision of the apical 40 pm (Lazof, 1991b). Other investigators have analysed net ion concentrations in shoot apices of salinized plants specifying segments of the order of 2-3 mm (e.g. Munns et al., 1988). B. TRANSPORT TO ZONES PROXIMAL TO THE MERISTEM IN POACEAE

The spatially defined elongation zone of S. bicolor leaves was dissected into 3 mm segments and analysed for Na-', K + , Mg2+and Ca", respectively (Bernstein et al.,

150

D.B. LAZOF and N. BERNSTEIN

1993b). The segment closest to the leaf base (0-3 mm from the point of attachment to the node) included the cell division zone of the LBM. This first segment had a relative elemental growth rate only 20% of that of the next 3 mm segment in both control and salt-stressed leaves, and only about 7% and 11% of the maximal relative elemental growth rates of control and salinized treatments, respectively. Four days after commencement of salinization (24h after the final salt level reached 100 mM NaCl) a reduction in [K], [Ca] and [MgJ and a 60-fold increase in “a] was reported as compared to plants grown with 1 mM NaCl. Although [K] and [Mg] were reduced 10% and 7%, [Ca] was reduced 75% (SAR = 63, Bernstein er al., 1995). This suggests that the CaZS supply to meristematic cells may be restricted by salinization. When the [Ca]med,un, was elevated from 2 to 10 mM, [Cal of control tissue closest to the node (including the cell division zone), increased fivefold, to attain the level in control tissue (Bernstein, unpublished).

VI.

PHLOEM TRANSPORT AND ION RECIRCULATION UNDER SALINITY

Another approach towards understanding the challenges to mineral transport for the meristematic zone, would be to consider effects on physiological processes, such as phloem transport, ion recirculation and xylem-phloem exchange, which are thought to be especially important in this transport. The recirculation, or retranslocation, of ions within the shoot of a plant is crucial to the maintenance of nutrient levels within growing tissues. This was noted even in the earliest studies of short-term nutrient studies, in which transpiration and xylem transport were shown to dictate the immediate tracer distribution which corresponded poorly to nutritional demand within the shoot (e.g. Stout and Hoagland, 1939). Movement against the water potential cannot occur through the xylem or the apoplast, so, at least over long distances, such as in export from mature transpiring leaves, phloem transport would be required. Xylem backflow out of major transpirational sinks on a diurnal cycle might also be important (Palzkill and Tibbitts, 1977; Wiebe ef ul., 1977; Tibbitts, 1979). Two distinct processes can be included under the concept of recirculation within the shoot. One process occurs on the time scale of organ development and can be adequately characterized as export during organ senescence, with an associated import into other aerial organs during organ formation. This process involves remobilization of nutrients from mature cells and tissues and occurs mainly from the leaf mesophyll of ageing and senescing leaves. A second process occurs on a much shorter time scale, within a few hours, or on a diurnal rhythm. This process may more accurately be characterized as xylem-phloem exchange within vascular tissue. It has sometimes been referred to as petiolar recirculation, rather than ‘mesophyllar’. This short-term process involves more of a continuous and circuitous mobilization of nutrients, rather than a ‘remobilization’.These two types of recirculation have not always been clearly distinguished in the literature. They

THE NaCl lNDUCED INHIBITION OF SHOOT GROWTH

15 1

do share several common features. Both processes may be largely regulated by the ability of a tissue to include or exclude specific ions from the phloem sieve tubes, both may contribute to nutrient supply to meristematic tissue and the impairment of either may be critically involved in the mechanism of NaC1-induced growth inhibition.

A.

REMOBILIZATION OF NUTRIENTS FROM AGEING SHOOT TISSUES, 'LONG-TERM RECIRCULATION'

There have been several studies of the exclusion of Na' from young shoot tissues by the selective remobilization of K ' (retention of Na '1 from ageing and senescing leaves. The underlying or, at times, explicit hypothesis has been that a genotype's increased capacity for selective K ' recirculation and Na' retention in the mature and senescing shoot tissues provides better protection to growing shoot tissues from 'ion excess' (section III.C.1). Even those studies restricted to Naf and K' remobilization implicitly include the idea that growth-related nutrient supply is site-specific and more complex than translocation from root to the shoot as a whole and are, therefore, relatively sophisticated. 1. Espc.rittienrrrtiol1it~iti~ti Using ii 'Pulsed' Liihellirig of the Roots Greenway (1962) showed that during five days in a '0 NaCl' nutrient solution (a 'chase') about 100 pmol of CI moved into the youngest leaves and apex of a barley shoot from older portions of the shoot. Meanwhile less than 10 pmol of Na' had recirculated into these portions of the plant, despite the fact that the [Nal and [Cll after the preceding 5-day salinization were each about 300 pmol (gDW,l,,,,t)--'. Under the same conditions, less than 200 pmol of "Na' moved into young leaves (numbers 5 and 6) during a 10-day chase, following 5 days of labelling with *"a ', although about ten times as much moved "Na' into the oldest leaf (from label in the root) during the same period (Greenway et ml., 1965). In a salt-sensitive dicot (Phcrsmlus i d g a r i s ) CI- partitioning to younger shoot tissue, under similar conditions of a chase following a 5-day labelling period, resulted in much greater portions of the whole plant Na' redistributing into the youngest leaves than had been evident with barley (nearly 20% after il 10-day chase, Greenway et al., 1966). These early studies demonstrated that on the time scale in which leaves proceed through their development, Na ' is relatively slow in recirculation. The later report intended to contrast regulation of Na ' transport in salt-sensitive and relatively salt-tolerant species; however, there were no recirculation experiments performed for the more tolerant species. There was also no evidence provided relevant to an impairment of recirculation processes by salinization (i.e. no non-inhibitory Na controls). In O r p r sativa, following a 48 h labelling period, 22Na+ continued to retranslocate at very low rates towards younger shoot tissue over a 10-day period (Ye0 and Flowers, 1982). The incremental increases in "Na' seemed to follow the

152

D. B. LAZOF and N. BERNSTEIN

changing distribution of mass within the shoot, so that perhaps much of the 22Nat was originating in the root of the plant and merely translocating according to the relative size of transpirational leaf surface. The distribution of transpirational leaf surface, of course, shifts towards later developing leaves through a long-term study. Recirculation of 22Nat, "CI- and 86Rbt was investigated in the halophyte Suaeda marifima under non-stressing salinity conditions (Yeo, 198 1). By applying the tracer for 5 days and then calculating the ratios of radioactivity between primary and axillary leaves, for axillary leaves which either had or had not been developed at the time of labelling, it was found that CI- was slightly more capable of recirculation than was Na'. The ratio of radioactivity in axillary to primary leaves for the axillary unformed at the time of root labelling was 35% greater for "C1than for 22Na' . Meanwhile *'Rb+ redistributed with equivalent radioactivity between the primary and unformed axillary leaves. Effects of a salinity-induced growth inhibition on recirculation were not considered. Wieneke and Lauchli (1980) used a similar approach with G. max, labelling the plants for 24 h in "Nat and then determining redistribution after 7 days in unlabelled solution. Total radioactivity of the apex had increased up to 75%during the chase, with three times as much 22Nat recirculation in the more salt-tolerant of two cultivars. Again, the effect of salt stress was not considered (no comparison with non-stressed controls). 2. Experiments Involving Adding Tracer to Mature Shoot Tissue Another approach to the investigation of recirculation within the shoot was used to make a preliminary assessment of Na' and C1- redistribution within the shoots of salinized plants for seven plant species, by application of "Na' and 36C1- to the apical portion of a developed leaf and assessing movement into younger, superior leaves after 48 h (Lessani and Marschner, 1978). The two species which showed the greatest capacity for recirculation of Na' were Capsicum annuum (pepper) and Carthamnus finctorius (safflower), but only C. annum was particularly saltsensitive. In these two species 77% as much '*Na+ was transported to the younger leaves compared with the basal portion of the leaf receiving the application, whereas this parameter averaged just 16% for the remaining five species. Among the seven species the more salt-sensitive species tended to recirculate less 3hCl to the shoot apex. Effects of salinization on shoot recirculation were not examined. 3. Experiments Determining Net Fluxes and Contents of Xylem and Phloem Long-term recirculation was studied in H. vulgare, L. albus and Leptochloa fusca by determination of the changes in organ ion content over the course of several days, often combined with determinations of xylem and phloem contents (Jeschke and Wolf, 1985; Jeschke et al., 1986; Munns et al., 1986; Wolf and Jeschke, 1987; Wolf et al., 1990; Jeschke et al., 1992, 1995). Similar to much of the work discussed above, these studies have generally shown that K* is selectively retranslocated out of ageing and senescing leaves whereas Na+ tends to be retained. This work, however, has also attempted to support several other concepts important

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

153

to an understanding of how recirculation interacts with salinity. For example, in one of the rare comparisons of the genotypic response of recirculation to salinity, it was suggested that the selectivity for K' in phloem loading (for leaf export) was much higher in the more salt-tolerant barley genotype (Jeschke and Wolf, 1985). The evidence for this, however, was not compelling. Although both the leaf content and [K] of young leaves were decreased by salinization this occurred to about the same extent in both genotypes (treatment with 100mM NaCI, SAR = 62) when compared to the 50 p M Na' control solution. Perhaps a higher non-inhibitory level of salinity would have led to a different result. The rates of leaf emergence for the cultivars were not shown for control conditions, hence the calculations of K' import of individual leaves on particular days could not be directly ascribed to a salinization effect. A later study including only the more salt-tolerant cultivar, analysed the contents of the xylem and phloem and indicated that Kf was selectively loaded into leaf phloem (and Na+ retained) and was then retranslocated out to the stem (Wolf and Jeschke, 1987). The selectivity of export from these well-developed leaves was insensitive to salinization, as raising the solution [Nalmedlum from 10 to 100 had no effect on the phloem exudation of K'. The portion of K.' supplied to the youngest leaf over a four-day period was little changed by salinization from 1 to l 0 0 m M with NaCl (17.6% vs. 12%), although growth and K' accumulation were greatly reduced. Still another report with this same barley cultivar showed that phloem transport of K' to a young, just emerged leaf was reduced from 46% to 25% of total K ' import into that leaf (from all shoot portions and the root) when the plants had been salinized to 100 mM NaCI, again compared to plants grown at 1 mM NaCl (Wolf et a/., 1990). The determinations were made during the first five-day period after emergence of that leaf and six days after the plants had been initially salinized. Xylem import to the same leaf during that period was reduced only 17%. The [Na]phloe,,, and [KIphlorlnof H. vulgrire was studied for 18 days of salinization (Munns et al., 1986). Recirculation of CI-, but not of Na', increased after 18 (but not after 10 days) as indicated by increased phloem levels in the highest salinity treatment (SAR = 116). Flux rates calculated from ion concentrations in exudates of xylem and phloem and volume fluxes indicated that recirculation via phloem was much greater for C1- than for Na'. However, even in the case of CI-, recirculation transport through the shoot phloem was less than 10% of the ion translocating upwards through the xylem. These latter data were produced only for the more moderate salinity treatment (Na:Ca of 25 and compared with a '0 Na' control). The effect of salinization on concentrations of ions and 18 organic components of phloem and xylem saps collected from stem portions of white L. albus was assessed by comparing the concentrations of these in plants growing with 1 or 40 mM NaCl for 15 days (Jeschke et a/., 1986). Results were not shown for specific petioles and stem segments and were not calculated as fluxes. An assumption that the levels of mineral ions in the phloem should represent relative rates of recirculation would be valid depending on the positions sampled in the shoots, assuming that, once in the phloem, the various components of phloem sap transport

154

D. B. LAZOF and N. BERNSTEIN

at similar long distance rates. Most likely in these 8-9-week-old plants the recirculating minerals were mostly being mobilized out of ageing leaves, although the precise developmental status of individual organs was uncertain. The petiolar phloem sap [K], [Ca] and [Mg] were reduced (by 21, 39 and 21%, respectively). whereas the [NaJ and [Cl] were, as one would expect, greatly increased ( I 1.5- and 8.6-fold, respectively) by salinization. The petiolar sap concentrations of SO4'- and Pi were also increased by the salinization. In a second report with L. albus recirculation was calculated after collection of xylem and phloem saps from particular petioles and measurement of net contents of shoot portions over a 10-day period after salinization from 0.05 to 40 mM NaCl (Jeschke et nl., 1992). The effect of salinization on phloem import to the terminal bud (inflorescence) were shown for C, N, K', Na' and CI-. Although the growth effects of salinization were not reported, the changes in C accumulation suggest that growth of the terminal inflorescence was unchanged, whereas that of the most apical leaves and petioles was reduced by about 50%. Total N and K accumulation to the terminal bud were each reduced by 25%. The portion of ten-day import which arrived via the phloem to the inflorescent bud of the salinized plants was 22% greater for N and reduced 39% for K'. The portion of Na-' and C1- supplied to the terminal bud by the phloem remained small (ca. 25%) despite 150- and 10-fold increases in the whole shoot accumulation of these elements after salinization. Over these ten days the portion of N uptake retranslocated again from the shoot was exactly the same in salinized and control plants (25%). Additional studies of phloem transport to young tissues in salinized Leptoclzloa fuscci are of dubious relevance to the present discussion, since the effects of salinization were not presented and it is unknown whether the plant was under any salt stress (Jeschke et ul., 1995).

B. XYLEMPHLOEM TRANSFER, 'SHORT-TERM RECIRCULATION'

In L. sativa recirculation of 32P was greatly inhibited by a moderate five day salinization as compared with control plants which were grown with 10 mM NaCl (Martinez et nl., 1996). The tiny stem of the lettuce plant was analysed as a part of the meristem base, but the rootlshoot interface was apparently discarded with no precise definition of where root and shoot were divided (see Lazof and Cheeseman, 1988a). During a 3 h 'chase' the 32P in the meristem base increased 230% and 130% for the control and salinized plants, respectively, during a chase and following a 3 h exposure in "P-labelled medium. At the same time, the amount of 3aP in the whole shoot increased by 108% and 10% for the two treatments. That 32P 'normally' undergoes very significant short-term redistribution in the shoot was evidenced by the increments of 32P moving into the various individual leaves of control plants during the 3 h chase. The leaf content of 32P increased less than 8% in the salinized plants during the 3 h chase, rather than the >loo% increase in the same leaves of plants grown at a non-stressing control level of NaCI. Although the youngest leaves doubled their 32Pcontent during the chase, the 32Pcontent of older

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

I55

leaves did not increase appreciably. Although '?P was moving also from the root during the chase, the dissimilarity in distribution during the labelling and chase periods must have been due to short-term recirculation from older to younger leaves. Exact estimates of immediate arrival and extent of redistribution will require more detailed time courses of translocation during both pulse and chase periods. It was also shown that transport from the root to the shoot during the chase was also less than 10% of that found in the control plants. Also in moderately salt-stressed cotton (SAR = 140) 3 h recirculation of 32Ptowards the apex (into two leaves weighing only 130 mg together and accounting for 7.5% of the shoot mass) was not reduced in salinized plants. although a more extreme salt treatment (SAR of 300) did cause a 55% reduction in "P recirculation (Martinez and Lauchli, 1991).

C. CALCIUM RECIRCULATION IN THE SHOOT

Calcium is the major nutrient most restricted from phloem transport and, therefore most likely to be critically affected on any inhibition of recirculation. In the past Ca' ' has been characterized as non-recirculating due to virtual immobility in the phloem (Hanson, 1984). However, it is clear that, although phloem immobility may be generally true for senescing leaves (e.g. Norton, 1963; Hill, 1980). even this is not absolute (see below). I n the present context, smaller exchangeable pools of Ca'~' associated with parenchyma cells of major and minor leaf veins and traces may be more crucial to short-term recirculation (Biddulph et nl., 1958; Biddulph and Nakayama, 1961; Norton, 1963; Millikan, 1967). Somehow, of course, even the most problematic of nutrients must be continuously supplied to the dividing cells and tightly regulated in both these and the cells undergoing extension, although the latter do have capacity for storage of mineral surplus. Transport of Ca" in the phloem does occur, at least in some plants (see reviews Lauchli, 1972; Pate. 1975; Bangerth, 1979). This has been demonstrated directly in leaves of Avrnu (Ringoet and Sauer, 1968) and in the shoot of Phmeolus (Biddulph and Nakayama, 1961). It has also been shown indirectly in a number of other dicots (Bukovac and Wittwer, 1957; Jeschke and Pate, 1991a,b). Indeed in some plants the [Ca]phlmmhas been greater than [Ca] in the xylem (Pate and Sharkey, 1975). Transport of Ca' ' through the phloem may also be important in particular locations within the plant, particularly where the xylem cannot supply the nutrient requirement, including non-transpiring fruits, storage organs and meristematic and expanding tissue (Pate, 1975). In the first 4 days after leaf emergence phloem transport of Ca' was 80% as great as xylem transport into young leaves of Ricinirs communis, whereas 8 days later, there was no net increase of [Ca],,,, from phloem transport (Jeschke and Pate, 1991a). It is likely that the phloem is involved in acropetal Ca' ' transport in dicots, probably by receiving Ca' ' from the xylem in petiolar leaf traces and transporting Ca2 basipetally in the leaf and petiole, at least for a short distance. The nutrient might then either continue transport through the

156

D. B. LAZOF and N. BERNSTEIN

phloem or be transported back into acropetally flowing xylem once more for movement towards the apex, although in the latter case it would again have to circuit through the dominant transpirational sinks superior to the leaf from which it is exiting. Abundant xylem phloem transfer cells have been found in leaf traces, although direct evidence is lacking implicating these in Ca2’ transport (Pate, 1969). Strong arguments and a mechanism have been provided by which transport through the phloem can be explained for some ‘non-phloem-mobile’ micronutrients (Udo and Scholz, 1993). Application of exogenous chelators has also been shown to promote the retranslocation of Ca2+from older to younger leaves (Millikan, 1965). Although Ca2* may be the most likely candidate for restricted transport towards the meristem due to disruption of recirculation, some research has also suggested a role for Ca2” in the maintenance of recirculation capacity in general (Martinez and Lhchli, 1991). Backflow of Ca” through the xylem has also been suggested as a means by which Ca” recirculates out of the rapidly transpiring mature leaves towards the stem and up towards the apex (Martin, 1982). The driving force would probably be created by cell expansion. Studies have suggested that xylem backflow may operate on a diurnal schedule, with the transport out of the transpirational sinks in the dark and at increased humidity (Palzkill and Tibbitts, 1977; Wiebe er al., 1977; Bangerth, 1979; Tibbitts, 1979). Humidity increases the water potential of the mature leaves, so that less negative water potentials at the meristem would be effective in driving acropetal transport. Apparently a relative humidity of 30% might be adequate to allow this nightly flux (Bangerth, 1979). Despite the strong logical arguments for a possible effect of salinity on the recirculation of Ca” there are apparently no available pertinent data. The decrease in 40% of [Ca]phlue,n in petioles of L. albus (twice the decrease found for K or Mg) was discussed above (section 1V.D). This decrease in [Ca]p,llorm also occurred simultaneous to a threefold increase of [Ca] of the xylem. It is likely that ion levels in petiolar phloem sap reflect rates of transport out of leaves.

D. SUMMARY OF SALINIZATION A N D RECIRCULATION

Most long-term recirculation studies have been limited by consideration of only Na’ and C1- and by the absence of a non-stressing NaCl control treatment. Of the pulse+hase studies, the one case in which nutrient, K’ (“Rb+), transport was considered, indicated no effect of salinization (Yeo, 1981). Additionally, the one study which included a non-inhibitory NaCl control, indicated that salt tolerance might be associated with an increase in maintained recirculation towards the apex, not a decrease as would be expected on the basis of the ‘young tissue protection hypothesis’ (section IV.A, Wieneke and Lauchli, 1980). Results of applying tracer to older leaves also did not support the protection hypothesis. Of the five studies in H. vulgare using whole organ and xylem/phloem analysis, only one study presented unequivocal evidence indicating that recirculation might be restricted by saliniza-

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

157

tion with a large decrease in K+ import from recirculating pools after salinization from 1 to 100 mM with NaCl (Wolf et al., 1990). In the L. albus studies a decrease in recirculation within the shoot due to salinization was indicated and not only for K', but also for N and more strongly for Cazt and Mg2+ (Jeschke et al., 1986). Besides the logical increase in Na ' recirculation following a 40-fold increase in [Na],ne,,lum, the recirculation of SO:- and P, were also increased. Taken together the studies on long-term recirculation present conflicting and weak indications for either the hypothesis that recirculation capacity regulates protection of the youngest shoot tissue, or that salinization affects nutrient recirculation. The few short-temi recirculation studies which have been carried out under saline and control conditions have indicated a large effect on nutrient recirculation of salinization (Martinez and Lauchli, 1991 ; Martinez ef al., 1996). Unfortunately it appears that only P, has been considered thus far, although an inhibition of recirculation has been shown for both young leaves and towards the apical meristem. There are reasons to suspect that Ca' ' recirculation might be the most critically affected of any major nutrient by restricted recirculation under salinization.

VII. SALINIZATION AND SHOOT NUTRITION: SPECIFIC NUTRIENTS A. POTASSIUM

It is generally recognized that K ' uptake to the plant, and deposition in both growing and non-growing tissues is reduced by salinization. Whether any shoot tissue approaches a deficient, metabolically limiting level of K during salinity stress remains open (section 1.C, I.C.2). Potassium is highly mobile in both apoplast and symplast, as well as in both phloem and xylem with no special transport restrictions relative to meristematic tissues ever having been suggested. Despite these reservations about the importance of disturbed K nutrition, discussion of K ' transport has been central to the discussion in most sections of this review, largely due to the great body of salinity related research which has considered such effects and the value of comparison to other nutrients. B. CALCIUM

Many studies conducted with reasonable Na:Ca ratios in the solutions have suggested that Ca' ' uptake, translocation and distribution may be critically affected by salinity. Transport of CaZf towards meristematic cells and cells in the earliest phase of extension may be reduced by salinization (sections 1V.C V). This may in part be due to a relatively low phloem mobility and the consequent difficulties in recirculating Ca2+ from transpirationally dominant mature shoot tissues to weakly transpiring growing tissues (section VI). The challenge of Ca' ' transport towards the meristem is heightened by its 'phloem immobility' and symplastic restrictions

158

D. B . LAZOF and N. BERNSTEIN

(sections V, VI). To maintain a critical nutrient level in actively dividing cells the rate of supply must occur at a rate equal to the product of the average rate of cell division and the total [Ca] of the meristem (primary cell wall included). Assuming a 30 h cell doubling time and a meristematic volume of 16 pl,the 4 pmol ( g F W - ’ concentration (Figs 5 and 7 in Lazof, 1991b). the meristem of a rapidly growing lettuce plant would require a net supply of about 50 nmol day- I . C. MAGNESIUM

Magnesium has been discussed above mainly as a point of comparison to the nutrition Ca (section III.B.2). Less study has been made of the effects of salinization on Mg nutrition, however, than has been made on Ca nutrition. This may be more due to lack of an appropriate radioisotope than to any intrinsic lesser importance of an effect. Among other essential metabolic roles, Mg” is required in mitosis, specifically in microtubule assembly, at specific and well-regulated levels. This means that the supply of Mg2+ to meristematic tissues, must be carefully maintained if shoot growth is to be continuous. In most saline soils (and in seawater) the [Mg] is relatively high (at least sevcral millimolar). Nonetheless, several investigations have included consideration of salinity effects on Mg nutrition (e.g. Lazaroff and Pitman, 1966; Downton, 1978; Ashraf et al., 1986, 1990; Cramer and Spurr, 1986; Jeschke et al.. 1986; Lynch et a/., 1988; Ehret et al., 1990; Omelian and Epstein, 1991). Only a few studies considering Mg nutrition have contributed towards an improved understanding of nutritional effects within the zones of cell division and rapid cell extension (e.g. Aslam et al., 1986; Grieve and Maas, 1988; Wolf et a/., 1990; Jeschke et al., 1992; Bernstein et al., 1995). These were discussed above (section IV.D), where it was judged to be still unclear whether [Mg] was generally low in young shoot tissues of plants subjected to salinity. Magnesium is not generally considered to be particularly prone to restriction either in phloem or symplastic transport. The consistency of data pointing towards reduced [Ca] in young tissues in the absence of reduced [Mg] supports the concept that some component of the recirculation process may be most severely limited under saline conditions (section VI). D. PHOSPHORUS

There has been considerable study of NaC1-induced effects on P nutrition. Several laboratories have reported that [P]aho,l,can be greatly increased by salinity. However, it has been suggested that this ‘P toxicity’ effect may be an artefact of the unrealistically high solution levels of P used in such studies (e.g. Bernstein et d., 1974; Nieman and Clark, 1976; Grattan and Maas, 1984. 1985; Martinez and Lauchli, 1991; Martinez et al., 1996). This is supported also by studies in soils where salinization did not lead to increased (e.g. Francois et id., 1984). Exactly what the minimum solution level of P is, however, which can result in

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

1.59

excessive P levels in the shoot, or which can exacerbate the salt-induced inhibition of shoot growth is not clear. Often such levels have been submillimolar levels. For example, concentrations as low as 120, or 100 p M in soybean (Grattan and Maas, 1984, 1988) or 100 pM in L. lzrteus (Treeby and Steveninck. 1988) were found to produce excess P accumulation. Concentrations as low as 20 pM P were found to produce increases in [P]ahcx,i in Brnscica olemcea, with trends towards the same in L. sntivn, Daucus cnrotn and other B. olerncen (Bernstein rt al., 1974). There have also been studies of the effect of salinization on P nutrition indicating decreased P transport. Some of these were carried out at high [PImrdlum. In R. coninzunis growing with 1.3 mM Pi there was a decrease in Pi exudation from excised roots of 90-9596 (Jeschke and Wolf, 1988). In Z . mays the [PIrhoo,decreased 50% with 0.17 mM P in the solution (Maas and Grieve, 1987). In G. hir.vutum there was a decrease in 32P translocation to the shoot even when growing at I mM Pi (Martinez and LPuchli, 1991). Several laboratories have investigated salinization effects on [P],ll,,c,i with attention to a possible association with relative species or genotypic salt tolerance. The more salt-sensitive Glycine fomentella had a greater decrease in [P]sl,,~ot, both at lower levels of salinization and after shorter periods at high salinity (as short as one day) (Wilson CT a/., 1970b). Also, in a study of five Glycine species the most salt-tolerant species had an increase in [P]ledl of 5% to 7% (at two harvest dates), while four species with greater salt sensitivity averaged decreases in [PIl,,, of 7%. to 20% (Wilson rt al.. 1970a). However elsewhere, of 22 accessions of G. wigktii the four most salt tolerant had the lowest decreases in [P]slloL,, (Gates et d.,1970). In that study all accessions decreased in although grown with 1 mM PI.Four genotypes of C. i i z u clearly segregated into two accumulating high and two accumulating only moderate P in their leaves during salinization when grown at 0.3 mM IPInledlum (either 600 or 300 nrnol (gDW)-l, Grattan and Maas, 1984). The two with higher [P].IIo,,,were also 15% more inhibited in growth than was the latter at this highest [P]mcdiu,,,. In four other G. mar lines the two genotypes which accumulated high [P]~llo,,i, were also almost twice as inhibited in growth as the more sensitive lines (Grattan and Maas, 1985). However, at low IP]ll,edi,i,nthe more sensitive lines neither attained higher (PIshoo,nor exhibited greater inhibition. The most salt-sensitive 2. tnoys genotype also had the greatest increase in [P]hlloo,with 0.17 mM [P]llledlum (Maas and Grieve, 1987). whereas in Agropyron ehngarum the most tolerant lines accumulated the highest [P]shou,(Shannon, 1978). In both G. Iiirsutunz and L. sativm short-term transport of ."P to young tissues and shoot apices were reduced by salinization (Martinez and Liiuchli, 1991; Martinez ef d., 1996). Decreases in young tissue [PI (or that of recirculating P) can even occur while P accumulation has been increased in the whole shoot (Jeschke et al., 1986). Similarly, in L. s u f i w grown with P, at 0.09 mM, salinization led to increascd [P]l,,l in the bulk shoot and most of the leaves, but to reductions averaging 9% of in the three youngest leaves (Fig. 5). This was a modest decrease in the youngest leaves, especially considering the much greater decrease in [S]lcaf.These data suggest that there might be ;L specific effect of salinization on the supply of P to

160

D. B. LAZOF and N. BERNSTEIN

7

8

9

10

11

12

13

Leaf number (younger-

14

1

Fig. 5 . Concentrations of P and S in leaves numbered 7 to 14 of salinized L. surivu 18 days after transferring seedlings to solution culture. Treatments, means and errors are as for Fig. 2.

young shoot tissue (section V1.B. 1). Rather than contradicting the ‘P toxicity’ concept, it is possible that a NaCI-induced P deficiency in young shoot tissue is physiologically linked with excessive [P]hhOO,. Perhaps a localized P deficiency within the growing zones elicits a signal to the root calling for increased uptake of P or increased allocation to the shoot. And, perhaps, at least in the case of phosphorus nutrition, this regulation at the shoot meristematic zones is not affected by ‘toxic’ P levels in the largest transpirational sinks. In general, the results of salinity effects on total [P]ahoo,have been highly variable and will need a good deal of careful experimentation with consideration of effects in growing tissue specifically, before anything of substantial value can be derived relative to the shoot growth inhibition.

E. NITROGEN

There has been only modest consideration of a possible role for N in the salt-induced shoot growth inhibition. Many studies treating salinity effects on N uptake and metabolism have been focused on uptake effects at the root plasma membrane, following the hypothesis of protein release from the root plasma membrane by osmotic shock (e.g. Klobus er al., 1988). This hypothesis is not relevant to the focus of the present review on nutrition of the growing shoot. Other studies of plant N nutrition and salinity have focused on effects of particular N sources on the shoot growth inhibition and on whole plant N uptake. Apparently NH: nutrition, may enhance the shoot growth inhibition (Bourgeais-Chaillou and Perez-Alfocea, 1992; Speer et al., 1994; Speer and Kaiser, 1994),however this may sometimes be an effect of amendment with NH4CI in particular (Speer et al., 1994). Alternatively, the enhanced growth inhibition under NH; nutrition, may be related to the dark period recirculation of NH: to younger shoot tissues (Ouny at al.,

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

161

1996), or a specific salinity effect on recirculation (section VI). Although unrelated to salinity stress, a recent review of NO; effects on plant development may prove valuable to any reader pursuing an interest in N effects in young and meristematic shoot tissues (McIntyre, 1997). In the more salt sensitive of two Glycine species [N] of young leaves decreased much more than did the [N] of the same leaves in its more tolerant relative (Wilson et d . ,1970b). This occurred, however, only at the most severe level of salinization (160 mM NaCI). In another study of five GIycine species the most salt-tolerant species had a reduction in [N]plu,,t of 2 I %, which was greater than the reduction in any of the other five, which together averaged a 12% reduction (Wilson et al.. 1970a). Of 22 accessions of Glycine wighrii the [N] in the four most salt-sensitive decreased 47%, whereas the four most tolerant averaged only a 21% reduction after 2 weeks of salinization (SAR = 68, Gates ef id., 1966). In two Citrus species the more salt tolerant had only a 8% reduction in IN]lra,, whereas the more salt sensitive had 26% reduction after four weeks of salinization and this occurred despite the much greater reduced leaf mass (18% vs. 3%) of the more sensitive species ( 1: 1 NH: to NO; supply, Lea-Cox, 1993). Interestingly, in the new shoot tissue the IN1 was only reduced in the sensitive species ( 3 I %), whereas it increased in the more tolerant by 27%. In 2. riinys salinity had no effect on [NIlelr.,however in salt-sensitive varieties of T aestivum and H. iwlgare lower levels of NOj supply (2 mM) led to enhanced reductions of grain yield due to salinization (Bernstein et al., 1974). There is also some evidence in conflict with the concept of NaCl disturbed N nutrition. In six vegetable crops the [N],hr,l,,was not reduced by salinization, but actually increased for the lowest levels of N supply in four of the crops (Bernstein et d.,1974). Reductions in N supply to the youngest leaves and to the apex (30%and 32%) in moderately salinized L. d b u s were only proportional to the reduction in NO; uptake (Jeschke ef al., 1992). Under similar salinity conditions R. rnmmunis had exactly the same reduction in NO, uptake as salinized L. alhus (32%), however net N shoot accumulation was more than twice as reduced in Ricinus and this was even greater (with a 75% reduction) for NH: fed plants (Peuke et al., 1996). Unfortunately, the earlier work from this same laboratory with great detail on N flow in the shoot of salinized R. commurzis did not attempt to separate ontogenetic and salt treatment effects (Jeschke and Pate, 1991b, 1992). The paucity of work on N nutrition and salt-induced shoot growth inhibition is astonishing given the importance of N in growth and development. Certainly some of this poverty is due to the lack of an easily handled radioisotope. The use of I3N may be especially prohibitive for studies involving dissection of the shoot and isolation of the growing regions, however application of stable isotope "N techniques has been made with root dissection (e.g. Lazof ef ol., 1992) and this should be relatively straightforward to apply to the plant shoot. In what seems to have been the only study of salinity effects on N transport to very young shoot tissue, it was reported that a moderate salinization did not decrease N deposition rates in any region (5 mm zones) of a wheat leaf, although growth effects were uncertain (Hu and Schmidhalter, 1997).

162

D. 9. LAZOF and N. BERNSTEIN F. MICRONUTRIENTS

With the exception of iron, there has been slow progress in the understanding of micronutrient nutrition in recent years (Kochian, 1991). Very few studies of micronutrients have been related to environmental stresses, other than that of micronutrient levels. Some of the recent work with iron has highlighted the necessity for recirculation of the nutrient within the shoot for transport towards young tissues (e.g. Zhang et d.,1996). This work may imply some restriction of iron nutrition of young tissues, if the hypothesis of inhibited recirculation and phloem transport has merit (section VI). A report that supplemental Mn could ameliorate the NaCIinduced inhibition of growth in H. vu1,gm-e seems to be unique in suggesting that a salt-induced growth inhibition could be ameliorated by addition of a micronutrient (Cramer and Novak, 1992). The work was preceded by evidence that [MnIshoo,in H . vulgctre decreased due to salinization (Cramer el nl., 1991). However, both reports suffer from serious flaws. In the first place, there was no clear statistical difference shown for [Mn]a,,oo,even in the most extreme treatment (>300 Na:Ca), although a trend towards reduction of [Mnlahoo1 was apparent for 2 out of 7 sampling dates. In the more moderate salinization (125 mM NaCl with 10 mM Ca) there was even less indication of a trend for reduced [Mn]bhUI)l occurring at any level of salinization (Cramer et al., 1991). Secondly, beyond the results for [Mn],h,,,, only analysis of [Ca]sh,,,,land total cation concentration were presented. The more detailed work on salinity amelioration by Mn, was conducted with a moderate salinization and showed that the mass of salinized plants could be doubled with either a few micromolar Mn added to the nutrient solution, or a few millimolar added as a foliar application (Cramer er al., 1991). Reductions in plant mass due to salinization were still 68% and 6.5% (nutrient solution and foliar treatments, respectively) with the amended Mn, as opposed to reductions of 84% and 79% without amended Mn. Under the conditions of the later study 25% of the growth inhibition (biomass attained after 23 days) was recovered by amending with Mn. There have been a few reports that plants evolved for optimal growth in saline environments may also be especially effective in excluding heavy metals, including the micronutrients Cu, Ni and Zn, when these appear in soils at high concentration (Fernandes and Henriques, 199I ; Otte et al., 1993). Logically, there is some reason to suspect that there might be an association of enhanced micronutrient regulation with salt tolerance, since retranslocation in the shoot and mobility through the phloem may be both particularly affected by salinity and crucially restricted for several micronutrients (Fe, Cu. Zn and Mn, Bukovac and Wittwer, 1957).

VIII. THE STUDY OF NUTRIENT STATUS AND TRANSPORT ON THE MICROSCALE It has been argued in the preceding pages that immediate causes of growth inhibition are best sought within the minute tissue volumes in which growth

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

163

processes are most intensive. In the case of the disturbed nutrition hypothesis this would involve chemical analysis of nutrients and ions, and quantification of their deposition rates within precisely defined dividing and expanding zones, as well as compartmentation and transport studies such as net ion flux, isotope flux studies and recirculation studies. All of these would have to be adapted to the microscale. in order to associate alterations directly with effects o n growth. Even among researchers who have assumed that ‘ion excess’ (Na and CI levels) were key parameters in understanding salinization effects growth, it has long been recognized that compartmentation of ions was of prime importance, although the scarcity of appropriate methods has often been lamented. For example, Ye0 ( I98 I ) summarized that the only two existing methods for looking at ion compartinentation, at that time, were EPXMA and compartmental analysis by efflux. Only the former of these two methods inspires the least confidence and will be discussed in any detail. As for compartmental analysis by efflux, the assumptions and limitations have been detailed previously from mathematical and physical perspectives (Cheeseman, 1986). That critique contained precise determinations of the inherent statistical limitations, even under ideal conditions. Little serious defence of the method has appeared during the subsequent 10 years. Of greater interest are the emerging methods for the study of nutrient status and transport at the microscale. Two of these methods are discussed in some detail below. Kinematic growth analysis quantifies tissue growth intensities along the protile of elongating tissue, such as a growing monocot leaf. The method allows examination of: ( I ) correlations between localized intensities of tissue growth and growth inhibition under stress and ( 2 ) localized levels and deposition rates of nutrients and other factors suspected to take part in the growth inhibition or maintenance processes. Indirectly, the method can also suggest alterations in the compartmentation of elements. since cells become progressively dominated by the cytoplasmic component as sampling approaches the leaf base. Secondary ion mass spectrometry (SIMS) is an isotope sensitive microanalytical method which is being used to directly determine localized isotope enrichment patterns and so to investigate alterations in nutrient compartmentation and nutrient flux through tissue on a cellular scale. In this case the results are obtained through direct imaging (isotopes are released from the first few nanometres of surface) of freeze-dried cryosections following short-term labelling and freeze fixation.

A.

KINEMATIC GROWTH ANALYSIS A N D ELEMENTAL DEPOSITION RATES

The study of elemental distribution profiles of elements in growth zones of graminaceous leaves is a powerful method allowing discrimination of those alterations in ion composition which are most closely associated with growth. Several such studies have been discussed in earlier sections of this review (Bernstein et d., 1993a, 1995; Hu and Schmidhalter, 1997). Several other instances of applying the method to study the effects of environmental stress, or to study the

164

D. B. LAZOF and N. BERNSTEIN

deposition of factors other than minerals can also be found in the recent literature (e.g. Schnyder and Nelson, 1987, 1988; Beemster and Made, 1996; Dodd and Davies, 1996). The first step in such an analysis is a kinematic analysis of growth. Growth kinematics analysis (GKA) allows quantitative measurements of parameters reflecting cell division and cell expansion specifically in unidirectionally growing organs such as developing graminaceous leaves. Although the growth zone of the grass leaf is concealed inside the whorl of older leaf sheaths and therefore not readily accessible for direct measurements, it is well suited for the study of leaf growth processes because of the relatively simple organization of its elongation zone (Volenec and Nelson, 1981; Schnyder and Nelson, 1988) and its distinct location. The existence of a ‘development gradient’ in the graminaceous leaf has long been employed to advantage in physiological studies. Less often specified are the temporal aspects implied by the spatial patterns. If growth is steady, then the ‘growth trajectory’ (a plot of distance from the leaf basal meristem which a particle traverses over time) allows inference of the time course of developmental properties (Silk and Erickson, 1979; Gandar, 1983). These processes include the accumulation of energy and mineral resources. A deposition rate is a useful characterization that combines experimental measurements of the spatial distribution of growth velocity and resource concentrations within the leaf growth zone. The observed concentration of a substance in growing tissue is, of course, the result of several processes. Local metabolism and transport can increase concentration, whereas growth-associated water uptake (often called ‘growth dilution’) can decrease it. The relative contributions of deposition and growth to the concentration profile can be evaluated with a onedimensional form of the continuity equation of fluid dynamics (Silk and Erickson, 1979; Silk, 1984). This equation provides the basis for evaluation of physiological factors suspected to be important in growth maintenance and inhibition (i.e. organic osmolites, carbohydrates, proteins, wall-loosening enzymes, solutes, etc.). Studies of S. hicolor leaves have revealed that [K], [Mg] and [Ca] in the growth zones of salt-stressed leaves were lower than in leaves of control plants and so was their deposition rate (sections 1.V.A-1.V.C). This occurred despite the lower expansion rates and the longer period of time that an elongating cell remains within the elongation region. The approach, however, may not be conclusive enough to determine a role in affecting a specific growth process (cell division or extension), nor may it be applicable to dicots, nor universally to monocots. The technique does not yield information on cellular compartmentation (thus, ‘microscale’, rather than microanalytic). After completion of such a study it remains essential to investigate subcellular concentration profiles along the expanding region and how these may be altered by stress, before any conclusion can be drawn about inhibition of a growth process. Microlocalization of several mineral elements has been performed by collection of vacuolar sap of growing cells of a S. bicolor leaf throughout the elongation zone (N. Bernstein, D. Tomos and W. Fricke, unpublished). This was done using a modified pressure probe sampler (Malone et al., 1989), as earlier reported in

165

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

TABLE 111

Effect of salinization on unicellular vacuolar sap concentrations oj expanding cells in the Sorghum bicolor leaf growing zone Distance from leaf base (cm) 1.5

3.5

Element

Control

Salt

Control

Salt

Na CI K

36 t 8 I1 + 16

91 +- 18 141 ? 31 130 ? 23

35 2 10 12+ 1 93+ I

90 2 6 141 30 112 ? 42

9 6 2 10

+

Leaf 6 was sampled on the first day after emergence above the whorl of older leaf sheaths ( 5 days after salinization). Plants were grown in I or 100 m M NaCI.

non-growing leaf tissue (Fricke ef nl., 1996). Ion concentrations in subsamples of unicellular sap were then determined by EPXMA. Vacuolar sap concentration in two locations (1.5 and 3.5 cm from the leaf base) are presented in Table 111. The length of the elongation zone of sorghum leaves is 3.0 and 2.4cm in the non-stressed and salt-stressed tissue, respectively (Bernstein et al., 1993a). Cells located 3.5 cm from the base are therefore young cells which have just ceased elongation growth. At location 1.5 cm from the leaf base, local growth intensity (extension per unit length) is at its maximum with a 35% reduction in local growth rate induced by the salinization. Vacuolar "a], [Cl] and [K] increased with salinization at both locations (Table 111). The increase at the 1.5 cm location was similar to that at the end of the growing zone. Together with published results indicating that in the bulk tissue of the 1.5 cm zone [Na] and [K] were higher than at the terminus of the growth zone (Bernstein et nl., 1995). the vacuolar data suggest that cytoplasmic and/or cell wall concentrations Na and K may be lower in the zone of most intensive expansion. Conclusions regarding deposition of elements into specific cell compartments and the effects of changes in such deposition rates which might affect growth under salt stress, will require microanalysis, or sampling of cell compartments other than the vacuole. A final step in combining kinematic growth analysis. elemental distribution profiles, and cell compartmental data would be determination of cell compartmentation along the leaf profile (i.e. morphometric analysis). Digital images of cross-sections throughout the growing zone of salt stressed and control maize leaves were acquired and relative volume fractions for wall, cytoplasm and vacuole determined (Bernstein, Neves Piastun, Yeo, Flowers and Thorpe, unpublished). As expected, the relative volume fraction of the vacuoles increases between the same two developmental locations described above (Table IV). Salinization led to a decreased relative volume of the vacuole in both locations, the reduction being larger in the younger cells (16% vs. 9%), whereas the cytoplasmic and cell wall volume fraction both increased. Since the cells of the stressed leaf, mature closer to the leaf base than in control leaves and their

166

D. B. LAZOF and N. BERNSTEIN

TABLE IV Morphometric analysis of expanding Iecrf tissue of Z. mays

Distance from leaf base (cm) 2.3

I .3

Control .

Salt

Control

Salt

0.61 i- 0.03 0.39 ? 0.03 0.41 2 0.06

0.82 ? 0.01 O.l8? 0.01 0.34 ? 0.17

0.76 ? 0.01 0.24 2 0.01 0.36 ? 0.014

.-.-

0.73 -+ 0.02 Vacuole 0.27 ? 0.02 Cytoplasm Cell wall X lo3 0.38 2 0.02

Leaf was sampled 4 days after plants were salinized to 100 mM NaCl (salt), or left at 1 mM NaCl (control).Errors are standard errors of the mean (n = 30).

displacement from the base is slower, then at each location the salt-stressed cells are more developed. The reduced relative vacuolar fraction in the salt-stressed leaf is opposite to that which would be expected on the basis of development and certainly affects compartmentation of the tissue. B. MICRODISSECTION

Microdissection is an approach applicable to both dicots and monocots, including the less accessible growth zones such as the graminaceous SAM. A cryostat equipped with a trimming tool could be used to dissect and isolate structures of interest such as leaf primordia and shoot meristems of defined dimension. Any such operation should be conducted only with frozen samples and under a light microscope, possibly employing micromanipulators for the dissection. Combination of this approach with a strictly microanalytic approach would allow the combination of radioisotope techniques, cellular compartmentation, improved quantitation of microanalytic imaging methods. as well as isolation of small zones of interest for preparation towards microanalysis, which are later cryosectioned or cryofractured. The method suggested here should not be confused with relatively crude attempts at dissection performed without the aid of a microscope, on fresh, injured, hand-held tissues at room temperature with disregard for current microtechniques and cryological principles. Although such studies can be found in the recent literature, they are easily identified by the lack of precise definition, both developmentally and dimensionally, of what has been isolated and analysed, as well as by the absence of any details on the conditions during dissection. C. SPECIMEN PREPARATION CONSIDERATIONS

With any microanalytical technique, the method of tissue preparation is of paramount importance. Artefacts introduced from the redistribution of analytes invalidate any interpretation. Specimen preparation can also influence the limits of

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

167

spatial resolution, background noise levels, signal suppression and other factors depending on the particular microanalytical method. There is little basis for interpretation of intracellular localization in samples which are chemically fixed and embedded in plastic. Fortunately, reports of such methods for ion microlocalization research seem to be published only rarely. Partial chemical methods, however, continue to be used. including freeze substitution protocols. In this method, the tissue is freeze fixed, but cell and apoplastic water are replaced with organic solvent, while the tissue supposedly retains the ionic species of interest within the respective cellular compartments (e.g. Harvey et nl., 1981; Hajibagheri et nl., 1987; Flowers et al., 1991). Serious challenges to this idea have been detailed elsewhere (Morgan et al., 1975; Morgan, 1980; Chandra and Morrison, 1992; Zierold, 1992; Steizer and Lehman, 1993; Chandra and Morrison, 1995). Such preparation methods are still widely practised for ion microlocalization, despite the criticisms and controversies and the availability of more purely cryological alternatives. More complete explorations of the controversies and underlying principles can be found in the literature (Morgan, 1980; Echlin, 1992). Although a pure cryological preparation, determination in freeze dried tissue blocks is also of limited value. Protoplasm from adjacent cells dries against each cell wall making any determination of cellular compartmentation unlikely (Echlin and Taylor, 1986). Furthermore, depending on the particular method employed, the volume of specimenheam interaction might be quite large (section VII1.D). Both of these difficulties might be largely overcome by cryosectioning quench frozen specimens and freeze drying the cryosection (section VII1.D). Such an approach might be followed for either EPXMA or SIMS. for example. A far superior method for EPXMA analysis in almost every respect is the analysis of frozen hydrated specimens, especially with regard to the virtual elimination of any opportunities for analyte redistribution. D. ELECTRON PROBE X-RAY MICROANALYSIS (EPXMA)

EPXMA has been used to monitor nutrient levels in meristematic cells and in young leaves (Storey et af.,1983; Seeniann and Critchley, 1985; Lazofand Lauchli, 199lb). Although such investigation is still infrequent, EPXMA undoubtedly has valuable contributions to make in investigating nutrient relations of young and meristematic tissue. The chief advantages of the technique, especially as practised with frozen hydrated cryofractured (FHC) samples, are that with proper standardization it allows quantitative estimates of nutrient concentrations on a microscale, at times allowing subcellular compartments to be analysed directly. Together with proper quench freezing, cryofracturing and transfer equipment the FHC EPXMA system allows analysis of tissue preserved nearly in the in v i w state, although several crucial limitations need be kept in mind. Chief among these is the fact that EPXMA is not strictly a surface analytical method, although the exact depth of analysis will vary with the substrate density and accelerating voltage of the primary beam.

168

D. B . LAZOF and N. BERNSTEIN

The volume of specimenheam interaction (the volume of tissue from which the characteristic elemental signal originates) for EPXMA is approximated by a teardrop 2 M O pm in diameter in a block of freeze-dried plant tissue (Goldstein er al., 1981; Lazof et al., 1996a). The nanometre resolution so impressive in the secondary electron image (SEI) bears only an imprecise relation to the size of dots an ‘X-ray dot map’, or the pattern dots superimposed over the SEI. Improvement in lateral resolution results when the samples are analysed frozen hydrated, rather than freeze dried, as increased specimen density leads to both a shallower penetration of the primary electron beam and a much shorter distance over which characteristic X-rays can escape from the sample (tenfold improvement in resolution, Boekestein and Stolo, 1980; Echlin and Taylor, 1986). The greatest lateral resolution in EPXMA can be obtained with ultrathin sections and when these remain frozen hydrated for analysis artefactual redistribution can also be avoided. However, obtaining ultrathin botanical cryosections remains impracticable (Michel and Hillmann, 1990; Stelzer and Lehman, 1993). A second major limitation of EPXMA, especially with the superior preparation, FHC EPXMA, is that concentrations must usually be in the millimolar range for any estimation of concentration to be made (Lazof and Lauchli, 1991a). This high detection limit means that use of an elemental analogue as a tracer (e.g. S?‘ for Ca”), although theoretically allowing detection by EPXMA, will not provide sufficient instrumental sensitivity for short-term studies. Furthermore, for particular elements there may be severe peak overlaps in EDS EPXMA. Of particular importance with regard to the material of this review, a severe peak overlap occurs between the major Ca peak (Ca ka) and the secondary K peak (K kp, Lazof and Lauchli, 1991 #2038). Although spectra can be corrected for the peak overlap, this would be more difficult for a characteristic X-ray map, requiring correction at each pixel (Lazof and Lauchli, 1991a). Use of a wavelength dispersive detector may also obviate this difficulty. With respect to sensitivity there can be great advantage in use of freeze-dried specimens with the resultant sampling of much greater tissue volumes, possibly complementing freeze-dried analysis to FHC EPXMA (Lazof and Lauchli, 1991a). Many FHC EPXMA studies with relevance to salinity, but irrelevant to the issue of shoot growth zones, have not been included in this review. The interested reader might begin with the review by Stelzer and Lehman (1993).

E.

SECONDARY ION MASS SPECTROMETRY (SIMS)

Biological applications of SIMS are recent, as the technique has been mainly limited to applications in materials sciences. Active programs in biological SIMS exist in France, Germany, Japan and the US. Both of the US laboratories with on-going active in situ biological SIMS programs insist on cryological preparation and exclude even freeze-substitution as inappropriate for preparing specimens to be analysed at the high spatial resolution and sensitivity of which SIMS is capable (Chandra et al., 1992b; Lazof et al., 1994b). The major disadvantage of using

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

169

freeze-dried cryosections is the opportunity for subcellular redistribution during freeze-drying. however movement of solutes and protoplasm will be primarily downward against the underlying substrate for sections of several micrometre fresh thickness (Waisel et nl., 1970; Lazof et al., 1994b, 1996a,c). Recently, SIMS has been applied to localization studies of aluminium in plant roots (Lazof et al., 1994a,b, 1996c) and to nutrient tracer studies in plant physiology (Goldsmith et al., 1996). Sophisticated short-term SIMS experiments involving 44Ca'+ labelling of either animal cell culture, or whole organ within a living organism have been carried out (Chandra et al., 1990, 1992a). Doublelabelling SIMS experiments with "Ca2"' and r6Mg2' have been conducted with intact roots of corn (Plate 2), and additional technical details can be found in recent reviews (e.g. Chandra and Morrison, 1995; Lazof er nl., 1995, 1996a). Transport of Ca". and Mg2* was followed in 400 pm longitudinal root sections of corn. Intact roots were exposed to the tracers 44Ca2+and 26Mg2' for times ranging from 0.5 to 5.5 h. Directly determined isotope distributions are shown in Plate 2 for the tracers and the two naturally dominant isotopes, 40Ca2+and 24Mg2t, following the 0.5 h exposure. Enrichment of the tracers after 30 min exposure to the tracer isotopes was quantitatively determined within defined regions of the 0.4 mm root tip from images acquired from replicate roots, and was found to be lowest in the most apical regions (Lazof, unpublished). Kinetic analysis of average enrichment in the 250 pm root tip demonstrated a more rapid exchange of 44Ca2' than 2sMg2t <0.5 or > 3 h respectively, Fig. 6). The kinetic and distributional studies together suggested that 44Ca' was transported into the 400 pm tip from more basal regions of the root. Mass spectra collected in the SIMS instrument indicated that there were no serious limitations due to mass interferences during collection of the Ca and Mg isotope images (major potential interferences were separated by 0.03 atomic mass units). The j4Cari~and "MgZt were already enriched 30- and 2.5-fold, respectively, after 30 min in the tracer solution over levels in unlabelled depleted tissue. In cryofractured cell cultures exchangeable Ca pools could be resolved to Golgi (Chandra et al., 1994). These results demonstrate a powerful new approach to in siru elemental microanalysis and short-term nutrient transport studies at the tissue and cell level. The root images shown here were collected with the instrument in 'ion microprobe' rather than in the 'ion microscope' mode, although the latter was used earlier in A1 toxicity studies (see Lazof et al., 1996a). In this mode of operation the linear detection range for incoming mass signals is increased a further 4 orders of magnitude up to lo9 counts per second. The primary beam current can be turned up allowing detection of lower levels of isotope or trace metal, without resulting in an off-scale mass signal for more abundant isotopes being ratioed. Lateral resolution in ion microprobe mode is 0.5 pm, perhaps less, on a perfect sample, but in practice is largely a function of specimen preparation (Lazof et al., 3996b). Detection limits for Na, K, Rb, Ca and Mg are all favourable due to high ion yield and limited mass interferences. The five elements listed should be detectable in freeze-dried cryosections within the range of pmol (gFW)-', on the basis of published factors +

170

D. B. LAZOF and N. BERNSTEIN c

C

E

f

.-o 30 L

C

w

El0

a,

2 a, a

0

1

2

3

4

5

6

Time (hours) Fig. 6. Tinie dependency of average enrichment for 44Caand '"Mg in the apical 250 Fni of corn root tips, grown in a complete nutrient solution with Ca and Mg concentrations of 0.2 and 0.4 mM. respectively. The roots were depleted to less than 0.2% AE for both isotopes prior to the labelling period. The isotope composition for Ca and Mg was the only variation between growth, depletion and labelling solutions. The enrichment values were determined by SIMS.

of relative sensitivity and limits already determined in plant cryosections (Ramseyer and Morrison, 1983; Ausserer et al., 1989; Lazof et al., 1996a). The great potential SIMS applications in plant physiology have been assessed more broadly elsewhere (Lazof et ul,, 1992).

F. SOME OTHER MICROANALYTICAL TECHNIQUES

Fluorescence has been used for monitoring fluctuations in cytosolic and vacuolar Ca' ', The technique can be applied to thick sections of tissue in a confocal system, allowing in vivo analysis in some specimens. Apparently, there has not yet been a study of Ca2+ relations within shoot meristems, or very young shoot tissues using fluorescent techniques. A primary limitation of fluorescence relative to the issues under discussion is the inability of the technique to discriminate isotopes and therefore to shed any light on issues of short term in siru ion transport. Beyond this, there are limitations to confocal fluorescent microscopy: ( I ) the technique is rarely quantitative, since numerous assumptions about quenching of the fluorescent signal, competitive binding and background need to be made in each cell phase; ( 2 ) there are depth limitations and it is necessary for excising a tissue such as a shoot menstem (Sandison et id., 1994), whereas frozen specimens are unsuitable. Microautoradiography is another potentially very useful microanalytical technique. It has been only sparingly applied to major issues in plant physiology and apparently never to nutrient transport to shoot meristems. This is probably due to the difficulty and time-consuming nature of the method. Certainly, excellent microautoradiography has been carried out on plant tissues (e.g. "Ca' , Luttge and

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

171

Weigl, 1962). It should be noted, however, that even though 45Ca2+is a relatively good isotope for this purpose the average track length is 230 pm through Ilford G5 (Lazof et al., 1992). Double-labelling experiments are extremely difficult, worse yet are studies of elements which do not have suitable radioisotopes (e.g. Mg).

IX. SUMMARY AND FUTURE PROSPECTS A.

REASSESSMENT OF CURRENT STATUS

Out of necessity physiologists and breeders tend to cast the most favourable light on the practical achievements and outcomes of their work. In a recent review of breeding for salt tolerance it was claimed that ‘remarkable achievements have been made in improving different agronomic traits through artificial selection during the past’ and that with regard to salt tolerance, in particular there has been great achievement in the breeding of alfalfa, barley, rice, pearl millet, maize, sorghum and other grasses (Ashraf, 1994). The optimism expressed in this particular review was not unusual. However, a greater measure of scepticism might be more helpful in providing a framework for future research needs. In examining the six crops and 17 improved lines in this review little evidence was found of remarkable achievements (Table V). Admittedly a multitude of criteria and methods have been used in the current literature to evaluate salt tolerance and the measure used here would not be universally the most relevant. However, the inhibition of vegetative shoot growth is certainly one valid measure and usually results in increased seed production for grain crops. Critical assessments need to be carried out with regard to additional criteria. If anyone working in salinity research were to take a few dozen salinity research articles from their files more or less randomly (as we have done) they would find that in the introductions, at least 75% mention the necessity for research based on the encroaching salinization of agricultural land or the increasing scarcity of high quality water for irrigation. This framework, however, is rarely applied to evaluation of the significance of breeding programmes. For only two of three legume crops was there even a minor improvement in the range of salinity tolerance, whereas for the third, M. sarivu, there was a decrease when plotted as a percentage of control growth (Fig. 7, Ashraf et ul., 1986; Ashraf, 1994). For the ‘hypothetical crop’ a case is shown wherein selection has resulted in a significant increase in the range of salinity tolerated by the crop species (70% increase, from 83 to 142mM NaCl). Indeed greater variations have often been found between genotypes of a crop species, as for example that reported in wheat, where the increase in salinity level could be estimated as severalfold (Table V, Qureshi et ul., 1980). This variation, however, is usually found among existing cultivars and reflects ‘natural’ variation rather than any achievement of breeding. The data for the four grass species was similar (Ashraf, 1994). In order to reduce growth by 50% in the four lines selected as tolerant, an additional 28,38, 18 or 0 mM NaCl was added

172

D.B. LAZOF and N. BERNSTEIN TABLE V The improvement of salt-tolerance as reported in 13 articles for 10 crops

Crop

Reference

~

Dobrenz et al. (1983)

Alfafa

No

Noble et al. (1984)

Alfafa

No

Ashraf et al. (1987)

Alfafa, two clover spp., rape

No

Epstein and Norlyn

Barley

No

Akbar (1986)

Rice

No

Rana ( 1977)

Wheat

No

Francois et al. (1984)

Sorghum

Yes

Rush and Epstein (1981a)

Tomato

Yes

Qureshi et a/. (1980)

Wheat

Yes

Kingsbury and Epstein

Wheat

Yes

( 1977)

(1984)

Comments'

Range"

~

~

~~~

..

Improved line has 7.5% increase in forage over the unselected. Fls with 'some improvement' in numbers of leaves damaged at 250 m M NaCI. Fls with improvements of 62, 35, 57 and 30% over most sensitive individuals of parent generation. No growth data and no comparison of grain yield for those lines selected for Na-tolerance. No data comparing any lines in any terms relevant to extent of improvement. No growth data. Comparison of available cultivars only (not bred for salinity-tolerance). Comparison of two cultivars. Not result of selection programme for salt-tolerance. All lines show fruit production reduced by 50% at lowest salt treatment (vegetative growth not shown). All 'improved' lines show less fruit production than the unimproved salt-sensitive cultivar at this salinity level. Selections from existing cultivars, not bred for improved salt-tolerance. By extrapolation 30% reduction in growth varied from an EC of 38 to 15 1 mmhos/cm. Constant Na to Ca. All extreme levels of salinity save one. No testing of progeny. Most resistant line only inhibited 59% at EC = 10.6, two developed lines improved but at EC = 10.6 inhibited 59 and 90%.

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

173

TABLE V Contd. Reference

Crop

Range’

Al-Khatib er 01. (1993)

Alfafa

Yes

Ashraf and McNeilly

Corn

Yes

(1990)

Commentsh

F1 improved up to 30% in terms of salinity which could be tolerated at 50% growth inhibition. Selected line improved up to 20% in salinity tolerated at 50% growth inhibition.

Salt-tolerant lines of crops developed either through selection or breeding. Whether a quantitative comparison o f vegetative growth for selected and non-selected genotypes over a range of salinities was included. ’Additional comments critical to judging the extent of the improvement in salt-tolerance.

over the level of NaCl which had inhibited the non-selected lines by 50% (15, 30, 14 and 0% increases in salinity). By comparison the natural variation among crops presented in the same article was eightfold. Many reports concerned with crop improvement for salt tolerance specify establishing whether salt tolerance is heritable as their main objective. Certainly most of the cited reports have indeed demonstrated that heritability exists for many species and, thus, that future breeding would seem to hold promise. However, the ability to withstand 40 rather than 30mM NaCl before a given level of growth reduction results may not much extend the cropping range or ability to use low quality irrigation water. All levels of inhibition do not serve equally well for comparison. Differences in maintaining high production (say 70% control growth) is more important than the point at which a genotype might decline from 80% inhibition to 95%. Although our analysis of the literature in this respect is incomplete, it suggests that if the literature were critically reviewed, then even after decades of salinity research one might continue to wonder about any extension in crop range with respect to salinity. B. MODEL SYSTEMS

All four general hypotheses (section LB), with the possible exception of the turgor/osmoregulation hypothesis, need to be considered within complex tissues and morphologies and within minute volumes of undifferentiated, rapidly dividing cells. Although each hypothesis suggests a particular mechanism for salinity induced growth inhibition, they involve integrated synthesis, partitioning, longdistance transport and regulation of metabolism. The processes are organismal and poorly modelled as ‘average’ cellular accumulations within a whole plant or whole shoot. If the osmoregulatory/turgor hypothesis is not of major major importance in genotypic salt tolerance (section I.B.2) and if the ‘ion excess’ hypothesis is to be

D.B. LAZOF and N. BERNSTEIN

174 I

'

I

'

I

~

I

*

I

'

I

.

Trifolium alexandrinum

60

-

= 40

-

E

+

a-,

-

Q

c 20-

Selected

0

'

I

'

-

-.

__

0 Non-selected

Hypothetical crop -

Trifolium pratense

60

I

Medicago sativa

-

40-

20

-

1 I

0

,

I

50

,

I

100

,

I

150

"a]

,

,I

, 1 1 , , 1 , , 1

0

I

I , , , , , , .I. , . I

50

. . , . * I ,

100

!.I

I

.

,

,

,

,

,

150

mot m-3

Fig. 7. The effect of salinization on growth of two lines of each of four crop species, one of each selected for salt tolerance. Data are replotted for three legumes as percentage change from the non-selected line (replotted from Ashraf et al., 1987). Also shown are data for a hypothetical crop which has been improved for salinity tolerance by 7 1%, measured as the level of salinity that can be tolerated while maintaining 70% of control growth. Dashed lines show differences in salinity that can be tolerated by the two lines of each species while maintaining 70% of control growth.

questioned (section I.C.1) in its most generalized form (applied to tissue status throughout the shoot), then the value of cell culture physiology and selection becomes highly suspect. The usefulness of testing salt tolerance of angiosperms in cultured undifferentiated cells (e.g. Blum, 1985; Niu et al., 1995; Kuznetsov and Shevyakova, 1997; Winicov and Shirzadegan, 1997) should be carefully reconsidered as a model for tolerance mechanisms. Similar recommendations have been made in previous reviews (Munns, 1993), apparently without any response, nor apparent effect on the course of research.

C. IN SITU ELEMENTAL AND ISOTOPIC ANALYSIS

Within almost every section of this review the dearth of relevant literature on the basic physiology of shoot growth and development as affected by salinization has been remarked upon. Studies of primordia formation, leaf initiation and direct analysis of cell division have not been conducted, despite a great deal of evidence that these are affected by salinity. Nutrient supply to growing leaf tissues

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

175

(elongating regions of grass leaves or entire very small dicot leaves has been poorly studied, only to be outdone in rarity by data on nutrition of the meristematic zones. Vague developmental definition of organs and tissues from which samples have been collected and broad categories such as ‘young leaves’ and ‘growing leaves’ also severely limit meaningful interpretation. Development and utilization of modem microscale and microanalytical methods will involve precise definition of leaf development and size, the study and analysis of minute structures, the necessity of linking elemental and isotopic analyses within cells which are rapidly dividing and extending. Such physiological study is, in turn, requisite to understanding the processes contributing to growth inhibitions from environmental stress.

ACKNOWLEDGEMENTS The SIMS application to plant physiology and the work presented were achieved in collaboration with J. G. Goldsmith, R. W. Linton and G. Gillen (respectively of the Department of Chemistry, University of South Carolina, Aiken, South Carolina, 29801, USA; the Chemistry Department, University of North Carolina, Chapel Hill, N Carolina 27599 3290, USA; and the Surface and Microanalysis Science Division, Bldg. 222, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, USA). Support for the microanalytic approaches to plant salinity has been supplied by USDA CRCR 87 1 2462 and USDA NRICGP 96 35100 3245.

REFERENCES Abel, G. H. and MacKenzie, A. J. (1964). Salt tolerance of soybean varieties (Glycine tnax L. Memll) during germination and later growth. Crop Science 4, 157-161. Ackerson, R. C. (1981). Osmoregulation in cotton in response to water stress. 11. Leaf carbohydrate status in relation to osmotic adjustment. Plant Physiology 67, 489493. Akbar, M. (1986). Breeding for soil stresses. /ti “Progress in Rainfed Lowland Rice” (A. K. Gupta ed.) pp. 263-272. International Rice Research Institute, Los Banos, Philippines. Alberico, G. J. (1993). Is the salt tolerance of maize related to sodium exclusion? 1. Preliminary screening of seven cultivars. Journal of Plant Nutrition 16, 2289-2303. Al-Khatib, M., McNeilly, T. and Collins, J. C. (1993). The potential of selection and breeding for improved salt tolerance in lucerne (Medicago sutiva L.). Euphytica 65, 43-5 1. Arif, H. and Tomos, A. D. (1995). Water relation parameters of mature epidermal cells of wheat leaf. In “Biology of Salt Tolerant Plants” (M. A. Khan and I. A. Ungar, eds) pp. 83-93. University of Karachi Press, Karachi, Pakistan. Ashraf, M. (1994). Breeding for salinity tolerance in plants. Critical Reviews in Plant Sciences 13, 1 7 4 2 . Ashraf, M. and McNeilly, T. (1990). Improvement of salt tolerance in maize by selection and breeding. Plant Breeding 104, 101-107. Ashraf, M., McNeilly, T. and Bradshaw, A. D. (1986). The response to NaCl and ionic

176

D. B. LAZOF and N. BERNSTEIN

content of selected salt-tolerant and normal lines of three legume forage species in sand culture. New Phytologist 104, 4 6 3 4 7 1. Ashraf, M., McNeilly, T. and Bradshaw, A. D. (1987). Selection and heritability of tolerance to sodium chloride in four forage species. Crop Science 26, 232-235. Ashraf, M., McNeilly, T. and Bradshaw, A. D. (1990). Patterns of ion distribution in selected NaCl tolerant and normal lines of four grass species. Biologia Plantarum 32, 302-3 12. Aslam, Z., Jeschke, W. D., Barrett-Lennard, E. G., Setter, T. L., Watkin, E. and Greenway, H. (1986). Effects of external NaCl on the growth of Atriplex amnicola and the ion relations and carbohydrate status of the leaves. Plant, Cell and Environment 9, 571-580. Aswathappa, N. and Bachelard, E. P. (1986). Ion regulation in the organs of Casuarina species differing in salt tolerance. Australian Journal of Plant Physiology 13, 5 33-545. Ausserer, W. A., Ling, Y.C. Chandra, S. and Morrison, G. H.(1989). Quantitative imaging of boron, calcium, magnesium, potassium and sodium distributions in cultured cells with ion microscopy. Analytical Chemistry 61, 2690-2695. Azhar, F. M. and McNeilly, T. (1987). Variation for salt tolerance in Sorghum bicolor (L.) Moench. under hydroponic conditions. Journal of Agronomy and Crop Science 159, 269-273. Ball, M. C. and Munns, R. (1992). Plant responses to salinity under elevated atmospheric concentrations of COz. Australian Journal of Botany 40, 5 15-525. Baluska, F., Kubica, S. and Hauskrecht, M. (1990). Postmitotic ‘isodiametric’ cell growth in the maize root apex. Planra 181, 269-274. Baluska, F., Barlow, P. W. and Kubica, S. (1994). Importance of the post-mitotic isodiametric growth (PIG) region for growth and development of roots. Plbnt and Soil 167, 3 1 4 1 . Bangerth, F. (1 979). Calcium-related physiological disorders of plants. Annual Review of P hytopathology 17, 97- 122. Bano, A. and Hayat, S. (1995). Effect of abscisic acid (ABA) on physiological and biochemical contents of Lens culinaris L. under saline conditions. In “Biology of Salt Tolerant Plants” (M. A. Khan and I. A. Ungar, eds) pp. 94-98. University of Karachi Press, Karachi, Pakistan. Banuelos, G. S., Zayed, A., Terry, N., Wu, L. Akohoue, S. and Zambrzuski, S. (1996). Accumulation of selenium by different plant species grown under increasing sodium and calcium chloride salinity. Plant and Soil 183, 49-59. Barlow, E. W. R. (1986). Water relations of expanding leaves. Australian Journal of Plant Physiology 13, 45-48. Barlow, E. W. R., Munns, R. E. and Brady, C. J. (1980). Drought responses of apical meristems. In “Adaptation of plants to water and high temperature stress’’ (P. J. K. Nc Turner, ed.) pp. 191-205. John Wiley, New York. Beemster, G. T. S. and Masle, J. (1996). Effects of soil resistance to root penetration on leaf expansion in wheat (Triticum aestivum L.): composition, number and size of epidermal cells in mature blades. Journal of Experimental Botany 47, 1651-1662. Benes, S. E. Aragules, R.,Grattan, S. R. and Austin, R. B. (1996). Foliar and root absorption of Na+ and CI- in maize and barley: implications for salt tolerance screening and the use of saline sprinkler irrigation. Plant and Soil 180, 75-86. Ben-Zioni, A,, Itai, C. and Vaadia, Y. (1967). Water and salt stresses, kinetin and protein synthesis in tobacco leaves. Plant Physiology 42, 361-365. Bernstein, L., Ehlig, C. F. and Clark, R. A. (1969). Effect of grape rootstocks on chloride accumulation in leaves. Journal o j the American Society of Horticulturul Science 94, 584-5 90.

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

I77

Bernstein, L., Francois, L. E. and Clark,R. A. (1974). Interactive effects of salinity and fertility on yields of grains and vegetables. Agronomy Journal 66, 412421. Bernstein, N., Lauchli, A. and Silk, W. K. (1993a). Kinematics and dynamics of sorghum (Sorghum bicolor L.)leaf development at various NdCa salinities. Plant Physiology 103, 1107-1114. Bernstein, N., Silk, W. K. and Lauchli, A. (1993b). Growth and development of sorghum leaves under conditions of NaCl stress. Planra 191, 433-439. Bernstein, N., Silk, W. K. and Lluchli, A. (1995). Growth and development of sorghum leaves under conditions of NaCl stress: possible role of some mineral elements in growth inhibition. PIanta 196, 699-705. Biddulph, 0. and Nakayama, F. S. (1961). Transpiration stream and ascension of calcium. Plant Physiology 6, 429-436. Biddulph, O., Biddulph, S. and Cory, R. (1958). Circulation patterns for phosphorus, sulfur and calcium in the bean plants. Plant Physiology 3, 293-300. Blum, A. (1985). Breeding crop varieties for stress environments. CRC Critical Review of Plant Science 2, 199-238. Boekestein, A. and Stolo, A. L. H. (1980). Quantitation in x-ray microanalysts of biological bulk specimens. Scanning Electron Microscopy II 321-334. Botella, M. A., Martinez, V., Pardines, J. and Cerda, A. (1997). Salinity induced potassium deficiency in maize plants. Journal of Plant Physiology 150, 200-205. Bourgeais-Chaillou, P. and Perez-Alfocea, F. ( 1992). Comparative effects of N-sources on growth and physiological responses of soyabean exposed to NaCl stress. Journal of Experimental Botany 43, 1225-1233. Breckle, S. W. (1995). How do halophytes overcome salinity? In “Biology of Salt Tolerant Plants” (M. A. Khan and I. A. Ungar, eds) pp. 199-213. University of Karachi Press, Karachi, Pakistan. Brouwer, R. (1963). The influence of the suction tension of the nutrient solutions on growth, transpiration and diffusion pressure deficit of bean leaves (Phaseolus vulgaris). Acta Botanicn Neerlandicn 12, 248-26 1. Bukovac, M. J. and Wittwer, S. H. (1957). Absorption and mobility of foliar applied nutrients. Plant Physiology 32, 428-435. Chandra, S. and Momson, G. H. (1992). Preparation of biological samples for ion microscopy. Pure and Applied Chemist7 64, 245-252. Chandra, S. and Morrison, G. H. (1995). Imaging ion and molecular transport at subcellular resolution by secondary ion mass spectrometry. International Journal of Mass Spectrometry and Ion Processes 143, 16 1-176. Chandra, S., Fullmer, C. S., Smith, C. A., Wasserman, R. H. and Momson, G. H. (1990). Ion microscopic imaging of calcium transport in the intestinal tissue of vitamin Ddeficient and vitamin D-replete chickens: a “Ca stable isotope study. Proceedings of the National Academy oj’ Sciences 87, 57 15-57 19. Chandra, S., Ausserer, W. A. and Morrison, G . H. (1992a). Subcellular imaging of calcium exchange in cultured cells with ion microscopy. Journal qf Cell Science 102, 417425. Chandra, S., Sod, E. W., Ausserer, W. A. and Morrison, G. H. (1992b). Preparation of biological samples for ion microscopy. Pure Applied Chemistry 64, 245-252. Chandra, S., Fewtrell, C., Millard, P. J., Sandison, D. R., Webb, W. W. and Morrison, G. H. (1994). Imaging total intracellular calcium and calcium influx and efflux in individual resting and stimulated tumor mast cells using ion microscopy. Journal of Biological Chemistry 269, 15186-15194. Chauhan, Y. S. (1987). Screening for tolerance to salinity and waterlogging, case studies with pigeonpea and chickpea. In “Proceedings of Consultant’s Workshop on Adaptation of Chickpea and Pigeonpea to Abiotic Stresses”, pp. 93-1 03. ICRI-SAT,

178

D. B. LAZOF and N. BERNSTEIN

Patancheru, Andra Pradesh, India. Cheeseman, J. M. (1986). Compartmental efflux analysis: an evaluation of the technique and its limitations. Plant Physiology 80, 1006-1011. Cheeseman, J. M. (1988). Mechanisms of salinity tolerance in plants. Plant Physiology 87, 547-550. Cheeseman, J. M. and Wickens, L. K. (1986). Control of Na’ and Kf transport in Spergularia marina In. Relationship between ion uptake and growth at moderate salinity. Physiologicu Plantarum 67, 15-22. Cheeseman, J. M., Bloebaum, P., Enkoji, C. and Wickens, L. K. (1985). Salinity tolerance in Spequlariu marina. Canadian Journal of Botany 63, 1762-1768. Clarkson, D. T. (1969). Metabolic aspects of aluminium toxicity and some possible mechanisms for resistance. In “British Ecological Society Symposium, no. 9” (I. H. Rorison, ed.) pp. 381-397. Blackwell Scientific Publishers, Oxford. Clarkson, D. T. (1993). Roots and the delivery of solutes to the xylem. Philosophical Transactions of the Royal Society of London 341, 5-17. Collier, G. F. and Huntington, V. C. (1983). The relationship between leaf growth calcium accumulation and distribution and tipburn development in field-grown butterhead lettuce. Scientiu Horticulturae 21, 123-1 28. Cramer, G., Epstein, E. and Lauchli, A. (1989). Na-Ca interactions in barley seedlings: relationship to ion transport and growth. Plant, Cell and Environment 12, 551-558. Cramer, G. R. (1992a). Kinetics of maize leaf elongation. 11. Responses of a Na-excluding cultivar and a Na-including cultivar to varying NdCa salinities. Journal of Experimental Botany 43, 857-864. Cramer, G. R. (1992b). Kinetics of maize leaf elongation. 111. Silver thiosulfate increases the yield threshold of salt-stressed plants, but ethylene is not involved. Hunt Physiology 100, 1044-1047.

Cramer, G. R. and Bowman, D. (1991a). Kinetics of maize leaf elongation. I. Increased yield threshold limits short-term, steady-state elongation rates after exposure to salinity. Journal of Experimental Botany 42, 1417-1426. Cramer, G. R. and Bowman, D. (I99 I b). Short-term leaf elongation kinetics of maize in response to salinity are independent of the root. Plant Physiology 95, 965-967. Cramer, G. R. and Novak, R. S. (1992). Supplemental manganese improves the relative growth, net assimilation and photosynthetic rates of salt-stressed barley. Physiologiu Platitarum 84, 600-605. Cramer, G . R. and Spurn, A. R. (1986). Salt responses of lettuce to salinity. 11. Effect of calcium on growth and mineral status. Journal offlant Nurrition 9, 131-142. Cramer, G. R.,Epstein, E. and Guchli, A. (1991). Effects of sodium, potassium and calcium on salt-stressed barley. 11. Elemental analysis. Physiologia Plantorum 81, 197-202. Cramer, G. R., Alberico, G . J. and Schmidt, C. (1994a). Leaf expansion limits dry matter accumulation of salt-stressed maize. Australian Journal of Plant Physiology 21. 663-674. Cramer, G. R., Alberico, G. J. and Schmidt, C. (1994b). Salt tolerance is not associated with the sodium accumulation of two maize hybrids. Australian Journal of Plant PhySiOlOgy 21, 675-692. Cuartero, J., Yeo, A. R. and Flowers, T. J. (1992). Selection of donors for salt-tolerance in tomato using physiological traits. New Phytologist 121, 63-69. Curtis, l? S. and Lauchli, A. (1985). Responses of kenaf to salt stress: germination and vegetative growth. Crop Science 25, 944-949. Dale, J. E. (1988). The control of leaf expansion. Annual Review of Plant Physiology 9. 267-295. Davenport, R. J., Reid, R. J. and Smith, F. A. (1997). Sodiumxalcium interactions in two wheat species differing in salinity tolerance. Physiologia Plantarum 99, 323-327.

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

I79

Delane, R., Greenway, H., Munns, R. and Gibbs, J. (1982). Ion concentratioii and carbohydrate status of the elongating leaf tissue of Hordeum vulgare growing at high external NaCI. Journal of Experimental Botany 3, 557-573. Dobrenz, A. K., Schonhorst, M. H., Stone, J. E., Thompson, R. K., Allen, S. and Smith, D. (1983). AZ-Germ salt-l nondormant alfafa germplasm. Crop Science 23, 807. Dodd, C. and Davies, W. J. (1996). The relationship between leaf growth and ABA accumulation in the grass leaf elongation zone. Plant, Cell and Environment 19, 1047-1056. Downton, W. J. S. (1978). Growth and flowering in salt-stressed avocado trees. Australian Journul ofAgricultura1 Reseurch 29, 523-534. Downton, W. J. S. (1984). Salt tolerance of food crops: prospectives for improvements. CRC Critical Review of Plant Science 1, 183-201. Dracup, M. (1991). Increasing salt tolerance of plants through cell culture requires greater understanding of tolerance mechanisms. Australian Journal of Plant Phj~siology18, 1-15.

Durand. M. and Lacan, D. (1994). Sodium partitioning within the shoot of soybean. Physiologia Plantarum 91, 65-7 1. Eaton, F. M. (1942). Toxicity and accumulation chloride and sulfate salts in plants. Journal of Agriculiural Research 64, 357-399. Echlin. P. (1992). “Low-Temperature Microscopy and Analysis”. Plenum Press, New York. Echlin, P. and Taylor, S. E. (1986). The preparation and x-ray microanalysis of bulk frozen hydrated vacuolate plant tissue. Journal of Microscopy 141, 329-348. Ehret, D. L.. Redmann, R. E. and Harvey, B. L. (1990). Salinity-induced calcium deficiencies in wheat and barley. Plant and Soil 128, 143-151. Elzam, 0. E. and Epstein, E. (1969). Salt relations of two grass species differing in salt tolerance I. Growth and salt content at different salt concentrations. Agrochim 13, 187-1 95. Epstein, E. and Norlyn, J. D. (1977). Seawater-based crop production: a feasibility study. Science 197, 249-25 I . Esau, K. (1977). “Anatomy of Seed Plants”. John Wiley, New York. Falkenmark, M. (1989). Water scarcity and food production. In “Food and Natural Resources” (C. W. H. D. Pimentel, ed.) pp. 164-191. Academic Press, San Diego, CA, USA. Fernandes, J. C. and Henriques, F. S. (1991). Biochemical, physiological and structural effects of excess copper in plants. Botanical Review 57, 246-273. Flowers, T. J. (1972). The effect of sodium chloride on enzyme activities from four halophyte species of Chenopodiaceae. Phytochemistry 11, 1881-1 886. Flowers, T. J. and Yeo, A. R. (1981). Variability in the resistance of sodium chloride salinity within rice (Ovza sativa L.) varieties. New Phytologisr 88, 363-373. Flowers, T. J., Troke, P F. and Yeo, A. R. (1977). The mechanism of salt tolerance in halophytes. Annual Review of Plani Physiology 28, 89-1 2 I . Flowers, T. J., Hajibagheri, M. A. and Yeo, A. R.(1991). Ion accumulation in the cell walls of rice plants growing under saline conditions: evidence for the Oertli hypothesis. Plant. Cell und Environment 14, 319-325. Fortmeier, R. (1995). Salt tolerance of maize ( E a mays L.): the role of sodium exclusion. Plant, Cell and Environment 18, 1041-1047. Francois, L. E., Donovan, T. and Maas, E. V. (1984). Salinity effects on seed yield, growth, and germination of grain sorghum. Agronomy Journal 76, 741-744. Fricke, W. ( 1 997). Cell turgor, osmotic pressure and water potential in the upper epidermis of barley leaves in relation to cell location and in response to NaCl and air humidity. Journal uf Experimental Botany 48, 45-58.

180

D.B. LAZOF and N. BERNSTEIN

Fricke, W., Leigh, R. A. and Tomos, A. D. (1996). The intercellular distribution of vacuolar solutes in the epidermis and mesophyll of barley leaves changes in response to NaCI. Journal of Experimental Botany 47, 1413-1426. Gandar, P. W. (1983). Growth in root apices. The kinematic description of growth and the rate of deformation. Botany Gazette 144, 1-19. Gates, C. T., Haydock, K. P. and Claringbold, P. J. (1966). Response to salinity in Glycinr. 2. Differences in cultivars of Glycine javanica in dry weight, nitrogen and water content. Australian Journal of Experimental Agriculture and Animal Husbandry 6, 374-379. Gates, C. T., Haydock, K. P. and Robins, M. F. (1970). Response to salinity in Glycine 4. Salt concentration and the content of phosphorus, potassium, sodium and chloride in cultivars of G. wightii. Australian Journal of Experimental Agriculture and Animal Husbandry 10, 99-1 10. Gauch, H. G. and Eaton, F. M. (1942). Effect of saline substrate on hourly levels of carbohydrates and inorganic constituents of barley plants. Plant Physiology 17, 347-365. Gleick, P. H. (1993). “Water in Crisis”. Oxford University Press, New York. Goldsmith, J. G., Lazof, D. B. and Linton, R. W. (1996). SIMS analysis of short-term double isotope labeling in whole plants. Secondary Ion Mass Spectrometry. In “Proceedings of the Tenth International Conference on Secondary Ion Mass Spectrometry”. John Wiley, New York. Goldstein, J. I., Newbury, D. E. Echlin, P., Joy, D. C., Fiori, C. and Lifshin, E. (1981). Practical techniques of x-ray analysis. In “Scanning Electron Microscopy and X-ray Microanalysis” (J. I. Goldstein, D. E. Newbury, P. Echlin, D. C. Joy, C. Fiori and E. Lifshin, eds) pp. 393-446. Plenum Press, New York. Gorham, J. and Bridges, J. (1995). Effects of calcium on growth and leaf ion concentrations of Gossypium hirsutum grown in saline hydroponic culture. Plant and Soirl76, 2 19-227. Grattan, S. R. and Maas, E. V. (1984). Interactive effects of salinity and substrate on phosphate on soybean. Agronomy Journal 76,668-676. Grattan, S. R. and Maas, E. V. (1985). Root control of leaf phosphorus and chlorine accumulation in soybean under salinity stress. Agronomy Journal 77,890-895. Grattan, S. R. and Maas, E. V. (1988). Effect of salinity on phosphate accumulation and injury in soybean I. Influence of CaCIJNaCI ratios. Plant and Soil 105,25-32. Greenway, H. (1962). Plant response to saline substrates. 11. Chloride, sodium, and potassium uptake and translocation in young plants of Hordeurn vulgare during and after a short sodium chloride treatment. Australian Journal of Biological Science 15, 39-57. Greenway, H. (1965). Plant response to saline substrates. VII. Growth and ion uptake throughout plant development in two varieties of Hordeum vulgare. Australian Journal of Biological Science 18,763-769. Greenway, H. and Osmond, C. B. (1972). Salt responses of enzymes from species differing in salt tolerance. Plant Physiology 49,256-259. Greenway, H. and Munns, R. (1980). Mechanisms of salt tolerance in nonhalophytes. Annual Review of Plant Physiology 1, 149-190. Greenway, H., Gunn, A., Pitman, M. G. and Thomas, D. A. (1965). Plant response to saline substrates. VI. Chloride, sodium, and potassium uptake and distribution within the plant during ontogenesis of Hordeum vulgare. Australian Journal of Biological Science 18,525-540. Greenway, H., Gunn, A. and Thomas, D. A. (1966). Plant response to saline substrates. VIII. Regulation of ion concentrations in salt-sensitive and halophytic species. Australian Journal of Biological Science 19,741-756.

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

181

Grieve, C. M. and Fujiyama, H. (1987). The response of two rice cultivars to external NdCa ratio. Plant und Soil 103, 245-250. Grieve, C. M. and Maas, E. V. (1988). Differential effects of sodiudcalcium ratio on sorghum genotypes. Crop Science 28, 659-665. Hajibagheri, M. A., Harvey, D. M. R. and Flowers, T. J. (1987). Quantitative ion distributions within root cells of salt sensitive and salt-tolerant maize varieties. New Phytologist 105, 367-379. Hajibagheri, M. A,, Yeo, A. R. and Flowers, T. J. (1985). Salt tolerance in Suaeda maritima (L.) Dum. Fine structure and ion concentrations in the apical region of roots. New Phytologist 99, 331-343. Hanson, J. B. (1984). The functions of calcium in plant nutrition. Advances in Plant Nutrition 1 149-208. Harvey, D. M. R., Hall, J. L., Flowers, T. J. and Kent, B. (1981). Quantitative ion localization within Suaeda maririma leaf mesophyll cells. Planta 151, 555-560. Hawkes, G. R., Luckhardt, R. L., McVickar, M. H. and Shaw, E. J. (1975). “Western Fertilizer Handbook”. Interstate Printers and Publishers Inc., Danville, Illinois. Hay, R. K. M. and Kemp, D. R. (1990). Primordium initiation at the stem apex as the primary event controlling plant development: preliminary evidence from wheat for the regulation of leaf development. Plant, Cell und Environment 13, 1005-1008. Hill, J. (1980). The remobilization of nutrients from leaves. Journal ofPZant Nutrition 2, 407444.

Hu, Y and Schmidhalter, U. (1997). Spatial distribution and net deposition rates of mineral elements in the elongating wheat (Triticwn aestivum L.) leaf under saline soil conditions. Planta 204, 2 12-2 19. Hussain, F. and Ilahi, 1. (1995). Effect of saline conditions on the germination, growth performance and oil composition of two cultivars of soybean. In “Biology of Salt Tolerant Plants” (M. A. Khan and I. A. Ungar, eds) pp. 31-42. University of Karachi Press, Karachi, Pakistan. Imamul Huq, S. M. and Larher, F. (1984). Osmoregulation in higher plants: effect of maintaining a constant Na:Ca ratio on the growth, ion balance and organic solute status of NaCl stressed cowpea (Vignu sinensis L.). Zeitschrifrfir Pflanzenphysiologie 113, 163-176. Jeschke, W. D. (1983). Cation fluxes in excised and intact roots in relation to specific and varietal differences. Plant and Soil 72, 197-2 12. Jeschke, W. D. (1984). K”-Na+ exchange at cellular membranes, intracellular compartmentation of cations, and salt tolerance. In “Salinity Tolerance in Plants” (R. C. Staples and G. H. Toeniessen, eds) pp. 37-66. John Wiley, New York. Jeschke, W. D. and Pate, J. S. (1991a). Cation and chloride partitioning through xylem and phloem within the whole plant of Ricinus conzmunis L. under conditions of salt stress. Journal of Experimental Botany 42, 1105-1 1 16. Jeschke, W.D. and Pate, J. S. (1991b). Ionic interactions of petiole and lamina during the life of a leaf of Castor bean (Ricinus communis L.) under moderately saline conditions. Journal of Experimental Botany 42, 1051-1064. Jeschke, W.D. and Pate, J. S. (1992). Temporal patterns of uptake, flow and utilization of nitrate, reduced nitrogen and carbon in a leaf of salt-treated castor bean (Ricinus communis L.). Journal of Experimental Botany 43, 393-402. Jeschke, W.D. and Wolf, 0. (1985). Na+ dependent net Kf retranslocation in leaves of Hordeum vulgare, cv. California Mariout and Hordeuni disrichion cv. Villa under salt stress. Journal of Plant Physiology 121, 21 1-223. Jeschke, W. D. and Wolf, 0. (1988). External potassium supply is not required for root growth in saline conditions: experiments with Ricinus communis L grown on a reciprocal split-root system. Journal of Experimental Botany 206, 1149-1 167.

182

D. B. LAZOF and N. BERNSTEIN

Jeschke, W. D., Pate, J. S. and Atkins, C. A. (1986). Effects of NaCl salinity on growth, development, ion transport and ion storage in white lupin (Lupinus albus. L. cv. Ultras). Journal of Plant Physiology 124, 257-274. Jeschke, W. D., Wolf, 0. and Hartung, W. (1992). Effect of NaCl salinity on flows and partitioning of C, N and mineral ions in whole plants of white lupin, Lupin albus L. Journal of Experimental Botany 43, 777-788. Jeschke, W. D., Klagges. S., Hilpert, A., Bhatti, A. S. and Sanvar, G.(1995). Partitioning and flows of ions and nutrients in salt-treated plants of Leptochloa fusca L. Kunth. I. Cations and chloride. New Phytologist 130, 23-35. Kahane, I. and Poljakoff-Mayber, A. (1968). Effect of substrate salinity on the ability for protein synthesis in pea roots. Plant Physiology 43, 1115-1 119. Kaiser, W. M. (1987). Effects of water deficit on photosynthetic capacity. Physiologiu Plantarum 71, 142-149. Kawasaki, T. and Moritsugu, M. (1978a). Effect of calcium on salt injury in plants I. Barley and rice. Berlin Report of Ohara Institute fur Landwirtschajiliche Biologie Okayamu Universitat 17, 72-8 1. Kawasaki, T. and Montsugu, M. (1978b). Effect of calcium on salt injury in plants. I. Maize and bean. Berlin Report of Ohara Institute fur Landwirtschajiliche Biologie Okayuma Universitat 17, 57-74. Kearney, T. H. and Cameron, F. K. (1 902). The effect upon seedling plants of certain components of alkali soils. U.S.D.A. Ofice of the Secretary Report 71, 1-40. Keamey, T. H. and Hatter, L. L. (1907). The comparative tolerance of various plants for the salts common in alkali soils. U.S.D.A. Plant Indusrty Bulletin 113, 7-22. Kent, L. M. and Lauchli, A. (1985). Germination and seedling growth of cotton: salinity-calcium interactions. Plant, Cell and Environment 8, 155-159. Kingsbury, R. W. and Epstein, E. (1984). Selection for salt-resistant spring wheat. Crop Science 24, 310- 315. Klobus, G., Ward, M. R. and Huffaker, R. C. (1988). Characteristics of injury and recovery of net no3 transport of barley seedlings from treatments of NaCI. Plant Physiology 87, 878-882. Kochian, L. V. (1991). Mechanisms of micronutrient uptake and translocation in plants. In: “Micronutrients in Agriculture”, 2nd edn (J. J. Mortvedt, F. R. Cox, L. M. Shuman and R. M.Welch, eds) pp. 229-296. Soil Science Society of America, Madison WI. Kramer, P. J. (1988). Changing concepts regarding plant water relations. Plant, Cell and Environment 11, 565-568. Kuznetsov, V. V. and Shevyakova, N. I. (1997). Stress responses of tobacco cells to high temperature and salinity. Proline accumulation and phosphorylation of polypeptides. Physiologia Plantarurn 100, 320-326. Lagerwerff, J. V. and Eagle, H. E. (1961). Osmotic and specific effects of excess salts on beans. Plant Physiology 36, 472-477. Lamoreaux, R. J. and Chaney, W. R. (1978). The plastochron index: a review after two decades of use. American Journal of Botany 65, 586-593. Lauchli, A. (1972). Translocation of inorganic solutes. Annual Review of Plant Physiology 23, 197-218. Lauchli, A. (1986). Responses and adaptations of plants to salinity. Acta Horticulturae, 190, 243-246. Lauchli, A. and Wieneke, J. (1979). Studies on growth and distribution of Na’, K+, and CIin soybean varieties differing in salt tolerance. Zeitschrifi fur die PJanzenErndhrung und Bodenkunde 142, 3-8. Layzell, D. B., Pate, J. S., Atkins, C. A. and Canvin, D. T.(1981). Partitioning of carbon and nitrogen and the nutrition of root and shot apex in a nodulated legume. Plant Physiology 67, 30-36.

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

183

Lazaroff, N. and Pitman, M. G. (1966). Calcium and magnesium uptake by barley seedlings. Australian Journal of Biological Science 19, 991-1005. Lazof, D. B. and Cheeseman, J. M. (1988a). Potassium compartmentation and transport across the roots of intact Spergularia marina Plant Physiology 88, 1274-1278. Lazof, D. B. and Cheeseman, J. M. (1988b). Sodium and potassium compartmentation and transport in the roots of intact lettuce plants. Plant Physiology 88, 1279-1284. Lazof, D. and Lauchli, A. (1991a). Complementary analysis of freeze-dried and frozen-hydrated tissue by electron probe x-ray microanalysis. Pluntu 184, 327333. Lazof, D. and Lauchli, A. (1991b). The nutritional status of the apical meristem of Luctuca sariva as affected by NaCl salinization: an electron-probe microanalytic study. Plantu 184, 334-342. Lazof, D., Rufty, T. W. and Redinbaugh, M. G. (1992). Localization of nitrate absorption and translocation within morphological regions of the corn root. Plant Physiology 100, 1251-1258. Lazof, D. B., Bernstein, N. and Lauchli, A. (1991). Growth and development of the Lnctuca sativa shoot as affected by NaCl stress: consideration of leaf developmental stages. Botanical Gazette 152, 72-76. Lazof, D., Linton, R. W. and Volk, R. J. (1992). The application of SIMS to nutrient tracer studies in plant physiology. Biology of the Cell 74, 127-134. Lazof, D. B., Goldsmith, J. G., Rufty, T. W. and Linton, R. W. (1994a). Rapid uptake of aluminum into cells of intact soybean root tips. A microanalytical study using secondary ion mass spectrometry. Plant Physiology 106, 1107-1 101. Lazof, D. B., Goldsmith, J. G., Suggs, C., Rufty, T. W. and Linton, R. W. (1994b). The preparation of cryosections from plant tissue: an alternative method appropriate for secondary ion mass spectrometry studies of nutrient tracers and trace metals. Journal of Microscopy 176. 99-109. Lazof, D. B., Goldsmith, J. G. and Linton, R. W. (1995). Applications of secondary ion mass spectrometry in plant biology. In “Applications of Modem Mass Spectrometry in Plant Science Research, Proceedings of the Phytochemical Society of Europe Vol. 40” (R. P. Newton and T. J. Walton, eds) pp. 81-112. Oxford Science Publications, Oxford, UK. Lazof, D. B., Goldsmith, J. G. and Linton, R. W. (1996a). The in situ analysis of intracellular aluminum in plants. In “Progress in Botany, Vol 58” (H. D. Behnke, U. Liittge, K. Esser, J. W. Kadereit and M. Runge, eds) pp. 112-149. Springer Verlag, New York. Lazof, D. B., Goldsmith, J. G. and Linton, R. W. (1996b). Ion transport in plants: nutritional and toxic metal SIMS studies. In “Metal Ions in Biology and Medicine, Volume 4. Proceedings of the Fourth International Symposium on Metal Ions in Biology and Medicine, John Libbey Eurotext, Montrouge, France” (C. P. J. Corbella, J. L. Domingo, J. C. Etienne and J. M. Llobet, eds) pp. 201-203. John Libbey Eurotext, Montrouge, France. Lazof, D. B., Goldsmith, J. C., Rufty, T. W. and Linton, R. W. (1996~).The early entry of aluminum into cells of intact soybean roots. A comparison of three developmental root regions using secondary ion mass spectrometry imaging. Plant Physiology 112, 1289-1300. Lea-Cox, J. D. (1993). Salinity reduces water use and nitrate-N use efficiency of citrus. Annuls of Botany 72, 47-54. Lechno, S.. Zamski, E. and Tel-Or, E. (1997). Salt stress-induced responses in cucumber plants. Journal of Plant Physiology 150, 206-2 1 I . Leidi, E. 0. and Sairz, J. F. (1997). Is salinity tolerance related to an accumulation in upland cotton (Gossypium hirsutum) seedlings? P!ant and Soil 190, 67-75.

184

D. B. LAZOF and N . BERNSTEIN

Lessani, H. and Marschner, H. (1978). Relation between salt tolerance and long distance transport of sodium and chloride in various crop species. Australian Journal of Plant Physiology 5, 27-37. Liu, J. and Zhu, J. K. (1997). Proline accumulation and salt-stress-induced gene expression in a salt-hypersensitive mutant of Arubidopsis. Plant Physiology 114, 591-596. Liittge, U. and Weigl, J. (1962). Microautoradiographical investigation of the uptan and transport of ’5S04 and 45Cain seedling roots of Zea mays and Pisum sativum. Planta 58, 113-126. Lynch, J . and Lauchli, A. (1985). Salt stress disturbs the calcium nutrition of barley (Hordeurn vulgare L.). New Phytologist 99, 345-354. Lynch, J., Thiel, G. and Lauchli, A. (1988). Effects of salinity on the extensibility and Ca availability in the expanding region of growing barley leaves. Botanica Acta 19, 355-361. Mats, E. V. and Grieve, C. M. (1987). Sodium-induced calcium deficiency in salt-stressed corn. Plant, Cell and Environment 10, 559-564. Maksymowych, R. (1973). “Analysis of Leaf Development”. Cambridge University Press, Cambridge, UK. Malone, M., Leigh, R. and Tomos. A. (1989). Extraction and analysis of sap from individual wheat leaf cells: the effect of sampling speed on the osmotic pressure of extracted sap. Plant, Cell and Environment 12, 919-926. Marschner, H., Kuiper, P. J. C. and Kylin, A. (1981a). Genotypic differences in the response of sugar beet plants to replacement of potassium by sodium. Physiologia Plantarum 51, 239-244. Marschner, H., Kylin, A. and Kuiper, P. J. C. (1981b). Differences in salt tolerance of three sugar beet genotypes. Physiologia Plantarum 51, 234-238. Martin, P. (1982). Stem xylem as a possible pathway for mineral retranslocation from senescing leaves to the eat in wheat. Australian Journal of Plant Physiology 9. 197-207. Martinez, V. and Lauchli, A. (1991). Phosphorus translocation in salt-stressed cotton. Physiologia Plantarum 83, 627-632. Martinez, V., Bernstein, N. and Lauchli, A. (1996). Salt-induced inhibition of phosphorus transport in lettuce plants. Physiologia Planturum 97, 118-122. Matsuda, K. and Riazi, A. (198 1). Stress-induced osmotic adjustment in growing regions of barley leaves. Plant Physiology 68, 571-576. Matyssek, R. and Maruyama, S. (1991). Growth-induced water potentials may mobilize internal water for growth. Plant, Cell and Environment 14, 917-923. McIntyre, G. I. (1997). The role of nitrate in the osmotic and nutritional control of plant development. Australian Journal of Plant Physiology 24, 103-1 18. Meyer, R. F. and Boyer, J. S. (1981). Osmoregulation, solute distribution, and growth in soybean seedlings having flow water potentials. Planta 151, 482439. Michel, M. and Hillmann, T. (1990). Cryosectioning of plant material frozen at high pressure. Journal of Microscopy 163, 3- 18. Michelina, V. A. and Boyer, J. S. (1982).Complete turgor maintenance at low water potentials in the elongating region of maize leaves. Plant Physiology 69, 1145- 1149. Millikan, C. R. (1965). Effects of chelation and certain cations on the mobility of foliar-applied 45Ca in stock, broad bean, peas, and subterranean clover, Australian Journal of Biological Science 18, 21 1-226. Millikan, C. R. (1967). Redistribution of 45Cain Trfolium subterratieurn L. and Antirrhinum majus L. Australian Journal of Biological Science 20, 1119-1 130. Morgan, A. J. (1980). Preparation of specimens: changes in chemical integrity. In “X-ray Microanalysis in Biology” (M.A. Hayat, ed.) pp. 65-165. University Park Press, Baltimore, MD.

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

185

Morgan, A. J., Davies, T. W. and Erasmus, D. A. (1975). Changes in the concentration and distribution of elements during electron microscope preparative procedures. Micron 6, 11-23. Munns, R. (1988). Why measure osmotic adjustment? Australian Journal of Plant Physiology 15, 717-726. Munns, R. (1993). Physiological processes limiting plant growth in saline soils: some dogmas and hypotheses. Plant, Cell and Environment 16, 15-24. Munns, R. and Cramer, G. R. (1996). Is coordination of leaf and root growth mediated by abscisic acid? Opinion. Plant and Soil 185, 3 3 4 9 . Munns, R. and Termaat, A. (1986). Whole-plant responses to salinity. Australian Journal of Plant Physiology 13, 143-160. Munns. R., Greenway, H., Delane, R. and Gibbs, J. (1982). Ion concentration and carbohydrate status of the elongating leaf tissue of Hordeum vulgare growing at high external NaCI. 11. Cause of the growth reduction. Journal of Experimental Botany 33, 574583. Munns, R., Fisher, D. B. and Tonnet, M. L. (1986). Na' and C1- transport in the phloem from leaves of NaC1-treated barley. Australian Journal of Plant Physiology 13, 757-766. Munns, R., Gardner, P. A., Tonnet. M. L. and Rawson, H. M. (1988). Growth and development in NaCl treated plants 11. Do Na' or CI- concentrations in dividing or expanding tissues determine growth in barley? Australian Journal of Plant Physiology 15, 529-540. Neuniann, P. (1997). Salinity resistance and plant growth revisited. Plant, Cell and Environment 20, 1193-1 198. Nieman, R. H. and Clark, R. A. (1976). Interactive effects of salinity and phosphorus nutrition on the concentrations of phosphate and phosphate esters in mature photosynthesizing corn leaves. Plant Physiology 57, 157-161. Niu, X., Bressan, R. A., Hasegawa, P. M. and Pardo, J. M. (1995). Ion homeostasis in NaCl stress environments. Plant Physiology 109, 735-742. Noble, C. L., Halloran, G. M. and West, D. W. (1984). Identification and selection for salt tolerance in Lucerne (Medicago sativa L.). Australian Journal of Agricultural Research 5, 239-252. Nonami, H. and Boyer, J. S. (1989). Turgor and growth at low water potential. Plant Physiology 89, 798-804. Nonami, H.. Wu, Y. and Boyer, J. S. (1997). Decreased growth-induced water potential. A primary cause of growth inhibition at low water potentials. Plant Physiology 114, 501-509. Norton, R. A. (1 963). Foliar and root absorption and distribution of phosphorus and calcium in the strawbeny. Proceedings of the American Society f o r Horticultural Science 82, 277-286. Oertli, J. J. (1966). Effect of external salt concentrations on water relations in plants. 11. Effect of the osmotic differential between external medium and xylem on water relations in the entire plant. Soil Science 102, 258-263. Oertli, J. J. (1968). Effects of external salt concentrations on water relation in plants. 5. Significance of external water potential and salt transport kinetics on rate of cell expansion. Soil Science 105, 216222. Omelian, J. A. and Epstein, E. (1991). Salt tolerance and ionic relations of wheat as affected by individual chromosomes of salt-tolerant Lophopyrum elongatum. Genome 34, 961-974. Otte, M. L., Haarsma, M. S., Broekman, R. A. and Rozema, J. (1993). Relation between heavy metal concentrations in salt marsh plants and soil. Environmental Pollution 82, 13-22.

186

D.B. LAZOF and N. BERNSTEIN

OUT, A,, Macduff, J. H., Prudhomme, M. P. and Boucaud, J. (1996). Diurnal variation in the simultaneous uptake and ‘sink’ allocation of NH; and NO; by Lolium perenne in flowing solution culture. Journal of Experimental Botany 47, 1853-1 853.

Palzkill, D. A. and Tibbitts, T. W. (1977). Evidence that root pressure flow is required for calcium transport to head leaves of cabbage. Plant Physiology 60, 854-856. Papp, J. C., Ball, M. C. and Terry, N. (1983). A comparative study of the effects of NaCl salinity on respiration, photosynthesis, and leaf extension growth in Beta vulgaris L (sugar beet). Plant, Cell and Environment 6, 675-677. Passera, C. and Albuzio, A. (1978). Effect of salinity on photosynthesis and photorespiration of two wheat species (Triticum durum cv. PEPE 2122 and Triticum aestivum cv. Marzotto). Canadian Journal of Botany 56, 12 1-1 26. Pate, J. S. (1969). Vascular transfer cells in angiosperm leaves. A taxonomic and morphological survey. Protoplasma 68, 135-156. Pate, J. S. (1975). Exchange of solutes between phloem and xylem and circulation in the whole plant. In “Transport in Plants. I. Phloem transport” (J. A. M. Mh Zimmermann, ed.) pp. 45 1-473. Springer, Berlin. Pate, J. S. and Sharkey, P. J. (1975). Xylem to phloem transfer of solutes in fruiting shoots of legumes, studied by a phloem bleeding technique. Planta 122, 11-26. Perera, L. K. R. R., Mansfield, T. A. and Malloch, A. J. C. (1994). Stomata1 responses to sodium ions in Aster tripolium: a new hypothesis to explain salinity regulation in above-ground tissues. Plant, Cell and Environment 17, 335-340. Perera, L. K. R. R., Silva, D. L. R. D. and Mansfield, T. A. (1997). Avoidance of sodium accumulation by the stomata1 guard cells of the halophyte Aster tripolium. Journal oj‘ Experimental Botany 48, 707-7 I 1. Peuke, A. D., Glaab, J., Kaiser, W. M. and Jeschke, W. D. (1996). The uptake and flow of C, N and ions between rots and shoots in Ricinus communis L. IV. Flow and metabolism of inorganic nitrogen and malate depending on nitrogen nutrition and salt treatment. Journal of Experimental Botany 47, 377-385. Pimentel, D., Houser, J., Preiss, E., White, 0..Fang, H., Mesnick, L., Barsky, T., Tariche, S., Schreck, J. and Alpert, S. (1997). Water resources: agriculture, the environment, and society. Bioscience 47, 97-106. Pitman, M. G. (1965). Transpiration and selective uptake of potassium by barley seedlings (Hordeum vulgare cv. Bolivia). Australian Journal of Biologicul Science 18, 987-998. Poethig, R. S. and Sussex, I. M. (1985a). The cellular parameters of leaf development in tobacco: a clonal analysis. Planta 165, 170-184. Poethig, R. S. and Sussex, I. M. (1985b). The developmental morphology and growth dynamics of the tobacco leaf. Planta 165, 158-169. Powell, M.J., Davies, M. S. and Francis, D. (1986). The influence of zinc on the cell cycle in the root meristem of a zinc-tolerant and a non-tolerant cultivar of Festuca rubra L. New Phytologist 102, 419428. Qureshi, R. H., Ahmad, R., Ilyas, M. and Aslam, Z. (1980). Screening of wheat (Triricum aestivum L.) for salt tolerance. Pakistani Journal of Agricultural Science 17, 19-26. Ramseyer, G. 0. and Morrison, G. H. (1983). Relative sensitivity factors of elements in quantitative secondary ion mass spectrometric analysis of biological reference materials. Analytical Chemistry 55, 1963-1 970. Rana, R. S. (1977). Wheat variability for tolerance to salt affected soils. In “Genetics and Wheat Improvement” (A. K. Gupta, ed.) pp. 180-184. Oxford & IBH Publishing Company, New Delhi, India. Rathert, G., Doering, H. W. and Witt, J. (1981). Influence of extreme K:Na ratios and high substrate salinity on plant metabolism of crops differing in salt tolerance. Journal of Plant Nutrition 4, 131-141.

THE NaCl INDUCED INHIBITION OF SHOOT GROWTH

187

Rauser, W. E. and Hanson, J. B. (1966). The metabolic status of nbonucleic acid in soybean roots exposed to saline media. Canadian Journul of Botany 44,759-776. Rawson, H. M. and Munns, R. (1984). Leaf expansion in sunflower as influenced by salinity and short-term changes in carbon fixation. Plnnt, Cell and Environment 7, 207-213. Rawson, H. M., Long, M. J. and Munns, R. (1988). Growth and development in NaCI-treated plants. I. Leaf Na and CI concentrations do not determine gas exchange of leaf blades in barley. Australiun Journal (?If’ Plant Physiology 15, 5 19-527. Reimann, C. ( 1993). Sodium relations in Chenopodiaceae: a comparative approach. Plant, Cell and Envimnnient 16, 323-328. Rengel, Z. (1992). The role of calcium in salt toxicity. Plant, Cell and Environment 15, 625433. Rhodes, P. R. and Matsuda, K. (1976). Water stress, rapid polyrobosome reductions and growth. Plant Physiology, 58, 63 1-635. Ringoet, A. and Sauer, G. (1968). Phloem transport of calcium in oat leaves. Plarztu 80, 15-20. Robinson, S. P., Downton, W. J. S. and Millhouse, J. A. (1983). Photosynthesis and ion content of leaves and isolated chloroplasts of salt-stressed spinach. Planr Physiologv 73, 238-242. Rozema, J., Rozema-Dijst, E., Freijsen, A. H. J. and Huber, J. J. L. (1978). Population differentiation with Festuca rubru L. with regard to soil salinity and soil water. Oecologia 34. 329-341. Rush, D. W. and Epstein, E. (1981a). Breeding and selection for salt tolerance by the incorporation of wild germplasm into a domestic tomato. Journul of the Anzerican Society of Horticulture Science 106, 699-704. Rush, D. W. and Epstein, E. (198lb). Comparative studies on the sodium, potassium, and chloride relations of a wild halophytic and a domestic salt-sensitive tomato species. Plant Physiology 68, 1308-13 13. Sachs. T. (1989). The development of vascular networks during leaf development. Plnnr Biochemistry and Physiology 8, 168-1 83. Sandison, D. R., Piston, D. W. and Webb, W. W. (1994). Background rejection and optimization of signal to noise in confocal microscopy. In “Three-Dimensional Confocal Microscopy: Volume Investigation of Biological Specimens”. (J. K. Stevens, L. R. Mills and J. E. Trogadis, eds) pp. 2 3 4 7 . Academic Press, New York. Schachtman, D. P., Bloom, A. J. and Dvorak, J. (1989). Salt-tolerant Triticurn X Lophopyrum derivatives limit the accumulation of sodium and chloride ions under saline stress. Plant Cell Environment 12, 47-55. Schnyder, H. and Nelson, C. J. (1987). Growth rates and carbohydrate fluxes within the elongation zone of tall fescue leaf blades. Plant Physiology 85, 548- 553. Schnyder, H. and Nelson, C. J. (1988). Diurnal growth of tall fescue leaf blades. Spatial distribution of growth, deposition of water, and assimilate import in the elongation zone. Plant Phvsiology 86, 1070-1076. Seemann, J. R. and Critchley, C. (1985). Effects of salt stress on the growth, ion content, stomata1 behaviour and photosynthetic capacity of a salt-sensitive species, Phaseolus vulgaris L. Planta. Shannon, M. C. (1978). Testing salt tolerance variability among tall wheatgrass lines. Agronomy Journal 10, 7 19-722. Silk, W. K. (1984). Quantitative descriptions of development. Annual Review of Planr Physiology 5, 479-5 18. Silk, W. K. and Erickson, R. 0. (1979). Kinematics of plant growth. Journul of Theoretical BiologJJ76, 48 1-50 1. Speer, M. and Kaiser, W. M. (1994). Replacement of nitrate by ammonium as the nitrogen

I88

D.3.LAZOF and N. BERNSTEIN

source increases the salt sensitivity of pea plants. 11. Inter and intracellular solute compartmentation in leaflets. Planr, Cell and Environment 17, 1223-123 1. Speer, M., Brune, A. and Kaiser, W. M. (1994). Replacement of nitrate by ammonium as the nitrogen source increases the salt sensitivity of pea plants. I. Ion concentrations in roots and leaves. Plant, Cell and Environment 17, 1215-1221. Stelzer, R. and Lehman, H. (1993). Recent developments in electron microscopical techniques for studying ion localization in plant cells. In “Plant Nutrition - from Genetic Engineering to Field Practice” (N. J. Barrow, ed.) pp. 3 5 4 5 . Kluwer Academic Publishers, Dordrecht, Netherlands. Storey, R., Pitman, M. G., Stelzer, R. and Carter, C. (1983). X-ray microanalysis of cells and cell compartments of Atriplex spongiosa I. Leaves. Journal of Experimental Botany 34, 718-794. Stout, P. R. and Hoagland, D. R. (1939). Upward and lateral movement of salt in certain plants as indicated by radioactive isotopes of potassium, sodium, and phosphorus absorbed by roots. Americun Journal of Botany 26, 320-324. Sunderland, N. and Brown, R. (1956). Distribution of growth in the apical region of the shoots of Lupinus albus. Journal of Experimental Botanv 7 , 126145. Taleisnik, E. and Grunberg, K. (1994). Ion balance in tomato cultivars differing in salt tolerance. I. Sodium and potassium accumulation and fluxes under moderate salinity. Physiologia Plantarurn 92, 528-534. Tennaat, A,, Passioura, J. B. and Munns, R. (1985). Shoot turgor does not limit shoot growth of NaC1-affected wheat and barley. Plant Physiology 77, 869-872. Tibbitts, T. W. (1979). Requirement for root-pressure flow to provide adequate calcium to low-transpiring tissue. Communications Soil Science Plant Analysis LO, 25 1-257. Treeby, M. T. and Steveninck, R. F. M. v. (1988). Effects of salinity and phosphate on ion distribution in lupin leaflets. Physiologia Planturum 73, 3 17-322. Udo, W. S. and Scholz, G. (1993). Nicotianamine: mediator of transport of iron and heavy metals in the phloem. Physiologia Planrarurn 88. 522-529. van Steveninck, R. F. M., Steveninck, M. E. v., Stelzer, R. and Lauchli, A. (1982). Studies on the distribution of Na and CI in two species of lupin (Lupinus luteus and Lupinus angustqolius) differing in salt tolerance. Physiologia Plantarum 56, 465473. Van Volkenburgh, E. and Boyer, J. S. (1985). Inhibitory effects of water deficit on maize leaf elongation. Plant Physiology 77, 190-194. Venables, A. V. and Wilkins, D. A. (1978). Salt tolerance in pasture grasses. New Phytologist 80, 6 13-622. Volenec, J. J. and Nelson, C. J. (1981). Cell cynamics in leaf meristems of contrasting tall fescue genotypes. Crop Science 21, 381-385. Waisel, Y.,Hoffen, A. and Eshel, A. (1970). The localization of aluminium in the cortex cells of bean and barley roots by x-ray microanalysts. Physiologia Plantarum 23, 15-79. Walker, R. R., Torokfalvy, E. and Downton, W. J. S. (1982). Photosynthetic responses of the citrus varieties Rangpur lime and Ertog Citron to salt treatment. Australian Journal of Plunt Physiology 9, 783-190. Wickens, L. and Cheeseman, J. M. (1988). Application of growth analysis to physiological studies involving environmental discontinuities. Physiologia Plantarum 73, 27 1217. Wiebe, H. J., Schatzler, H. P. and Kuhn, W. (1977). On the movement and distribution of calcium in white cabbage in dependence of the water status. Plant and Soil 48, 4 0 4 4 16. Wieneke, J. and Lauchli, A. (1980). Effects of salt stress on distribution of Na+ and some other cations in two soybean varieties differing in salt tolerance. Zeitschr$t fur die Pjlanzenernahrung und Bodenkunde 143, 55-61.

THE NaCl INDUCED INHIRITION OF SHOOT GROWTH

189

Wilson, J. R. (1967). Response to salinity in Glycine 3. Dry matter accumulation in three Australian species and G. javanicu. Australian Journal of Experimental Agriculture and Animal Husbandy 7 , 50-56. Wilson, J. R., Haydock, K. P. and Robins, M. F. (1970a). Changes in the chemical composition of three Australian species of G. wightii (G. juvanica) over a range of salinity stresses. Australian Journal qf Experimental Agriculture and Animal Husbandry 10, 156-165. Wilson, J. R., Haydock, K. P. and Robins, M. E (1970b). The development in time of stress effects in two species of Clycine differing in sensitivity to salt. Australian Journal of Biological Science 23, 537-55 I . Winicov, I. and Shirzadegan, M. (1997). Tissue specific modulation of salt inducible gene expression: callus versus whole plant response in salt tolerant alfalfa. Physiologiu Plantarum 100, 314-319. Wolf, 0. and Jeschke, W. D. (1987). Modeling of sodium and potassium flows via phloem and xylem in the shoot of salt-stressed barley. Journul of Plunt Physiology 128. 371-386. Wolf, O., Munns, R.,Tonnet, M. L. and Jeschke, W. D. (1990). Concentrations and transport of solutes in xylem and phloem along the leaf axis of NaCI-treated Hordeum vulgare. Journul of Experimental Botativ 41, 1 130- 1 141. Yeo, A. ( 1983); Salinity resistance: -physiologies and prices. Physiologia Planfururn 58, 2 14-222. Yeo, A. R. (1981). Salt tolerance in the halophyte Siiaedu mnritimu L. Dum.: Intracellular cornpartmentation of ions. Journul of‘ Experimental Botany 2. 487497. Yeo, A. R. and Flowers, T. J. (1982). Accumulation and localisation of sodium ions within the shoots of rice (Oryza sutivu) varieties differing in salinity resistance. Physiologiu Plantarum 56, 343-348. Yeo, A. R. and Flowers, T. J. ( 1985). The absence of an effect of the NdCa ratio on sodium chloride uptake by rice (Oryza sativa L.). New Phytologist 99, 81-90. Yeo, A. R. and Flowers, T. J. (1986). Ion transport in Suaeda maritima: its relation to growth and implications for the pathway of radial transport of ions across the root. Journal of Experimental B O ~ U F37, Z ~ J143- 159. Yeo, A. R. and Flowers, T. J. (1989). Selection for physiological characters - examples from breeding for salt tolerance. h i “Plants under Stress” (H. G . Jone, T. J. Flowers and M. B. Jones, eds) pp. 217-234. Cambridge University Press, New York. Yeo, A. R., Caporn, S. J. M. and Flowers, T. J. (1985). The effect of salinity upon photosynthesis in rice (OQW sutiiw L.): gas exchange by individual leaves in relation to their salt content. Joirrnul of Experirnenral Botany 36, 1240-1248. Yeo. A. R., Lee, K. S., Izard, P., Boursier, P. J. and Flowers, T. J. (1991). Short and long term effects of salinity on leaf growth in rice (0ry:u sativa L.). Journal of Experimental Botany 42, 88 1-889. Zhang, C., Romheld V. and Marschner, H. (1996). Effect of primary leaves on 59fe uptake by roots and 59fe distribution in the shoot of iron sufficient and iron deficient bean (Phaseolus vulgaris L.) plants. Plant and Soil 182, 75-8 1. Zhang, J. and Davies, W. J. (1990). Does ABA in the xylem control the rate of leaf growth in soil dried maize and sunflower plants’? Journal (?f E.xperimental Botany 41, 1 125-1 132. Zierold, K. ( 1992). Comparison of cryopreparation techniques for electron probi microanalysis of cells as exemplified by animal erythrocytes. Scanning Microscopy 6, 1137-1 145.

This Page Intentionally Left Blank

AUTHOR INDEX Numbers in italic refer to pages on which full references are listed

A Abdel-Aal, M. M., 38, 50 Abdel-Hay, E A.. 38, 50 Abel, G. H., 119, 128, 175 Ackerson, R. C., 118, 175 Adams, R. M., 32, 35, 49 Agrawal, M., 44, 45, 49 Ahmad, R., 115, 171, 172, 186 Ahmed, D. M., 37, 38,50 Akbar, M., 172, 175 Akohoue, S., 119, 176 Alberico, G. J., 116, 117, 118, 120, 121, 122, 116, 130, 132, 175, 178 Alexander, M., 20, 28 Alien, S., 172, 179 Al-Khatib, M.,173, 175 Allen, G.J., 79, 83, 89, 104 Alpert, S., 115, 186 Altschmied, L., 68, 72 Amthor, J. S., 33, 49 Amtmann, A., 80, 83, 84, 86, 87, 88, 91, 92, 95, 97, 98, 99, 100, 102, 103, 104 Amundsen, R. G., 41, 51 Anderson, J. A., 84, 85, 99, 104, IOS, I10 Andersson. M. E., 9, 10, 25 Andre, R., 37, 38, 50 Aniol, A., 8, 26 Aragules, R., 130, 176 Arif, H., 11 8, I75 Armstrong, F., 99, 104 Arnon, D. I., 9, 26 Ashenden, T. W., 18,28 Ashmore, M. R., 33, 34, 36, 39, 40, 42, 43, 49, 50, 51, 52 Ashraf, M., 115, 120, 129, 130, 131, 158, 171, 172, 173, 175, 176 Aslam, Z., 115, 117, 124, 133, 134, 136. 139, 140, 158, 171, 172,176. 186 Assmann, S. M., 98, 99, 102, 106. 108, 112

Astruc, S., 84, 108 Aswathappa, N., 134, 176 Atkins, C. A., 136, 152, 153, 157, 158, 159,182 Atkinson, C. J., 16, 17, 26, 29 Ausserer, W. A., 168, 169, 170, 176,I77 Austin, R. B., 130, 176 Ayah, F., 100, 104 Azhar, F.M., 115, 176 Azimov, R. R., 86, 104

B Bachelard, E. P., 134, 276 Baldwin, I. T., 20, 26 Ball, M. C., 118, 124, 176, 186 Ballarin-Denti, A., 97, 105 Baluska, F., 133, 176 Barnbawale, 0. M., 40, 41, 49 Bangerth, F., 155, 156, 176 Banno, H., 58, 73 Bano, A., 118, 123, 176 Banuelos, G. S., 119, 176 Barkla, B. J., 79, 104 Barlow, E. W. R., 118, 176 Barlow, P. W., 133, 176 Barrett-Lennard. E. G., 117, 124, 133, 134, 136, 139, 140, 158,176 Barsky, T.,115, 186 Bartlett, R. J., 6, 8, 26 Barz, W., 58, 71 Basset, M., 84, 108 Batschauer, A,, 56, 70, 71 Beaton, C. D., 8, 26 Becker, D., 84, 85, 98, 99, 105, 107, I09 Beemster, G. T. S., 164, 176 Bell, J. N. B., 39, 40, 42, 43, 46, 49, 50, 52 Benes, S. E., 130, 176 Bennett, A. B., 100, 110 Bennett, R. J., 6, 8, 26. 27 Benton, J., 36, 49

192

AUTHOR INDEX

Ben-Zioni, A., 126, 176 Berestovskii, G.N., 86, 104 Bernstein, L., 91, 105, 128, 158, 159, 161, 176, 177 Bemstein, N., 124, 134, 137, 138, 139, 140, 141-2, 144, 145, 147, 149, 150, 154, 157, 158, 159, 163, 164, 165, 177, 183, 184 Bertekap, R. L., 54, 73 Bertl, A., 85, 86, 87, 99, 102, 105, 110 Bhatti, A. S., 152, 154, 182 Biddulph, O., 155, 177

Biddulph, S . , 155, 177 Binzel, M., 78, 79, 100, I05 Blatt, M. R., 85, 87, 98, 99, 102, 104, 105, 109, 111 Block, A., 60, 72 Bloebaum, P., 120, 178 Bloom, A. J., 119, 120, 187 Blum, A., 174, 177 Blumwald, E., 79, 104, 105 Boekestein, A., 168, 177 Bogner, W., 17, 18, 26 Bonetti, A., 79, 105 Bonneaud, N., 84, 110 Bonte, J., 33, 49 Booth, C. E., 34, 51 Bosseux, C., 83, 84, 85, 98, 111 Bostock, R. M., 63, 70 Botella, M. A., 120, 127, 177 Boucaud, J., 160, 186 Bourgeais-Chaillou, P., 160, 177 Boursier, P. J., 125, 135, 189 Bowler, C., 61, 62, 63, 68, 69, 70, 73 Bowling, D. J. F., 78, 97, 105 Bowman, D., 118, 121, 178 Boyer, J. S., 118, 147, 184, 185, 188 Bracker, C. A., 64, 73 Bradley, D.J., 65, 70 Bradshaw, A. D., 120, 129, 130, 131, 171, 172, 175, 176 Brady, C. J., 118, I76 Brauer, D. K., 8, 30 Braun, Y., 79, 100. 105 Breckle, S . W., 120, 127, 177 Bregante, M.,85, 98, 107 Bressan, R. A., 64, 73, 76, 77, 78, 79, 100, 105, 109, 110, 174, 185 Bridges, J., 128, 180 Broekman, R. A., 162, 185 Brouwer, R., 125, 177 Brown, B., 56, 68, 69, 71

Brown, P. H., 163, 181 Brown, R., 125, 188 Brown, V., 34, 49 Brown, V. C., 46, 49 Brownlee, C., 14, 28 Briidem, A., 98, 100, 111 Brune, A., 160, 188 Brunet, J., 9, 10, 25 Buckel, S., 64, 73 Bukovac, M.J., 155, 162, 177 Busch, H., 84, 85, 98, 99, 105. 107, 109 Bush, D. R., 81, 105 Bush, D. S., 14, 16, 26, 85, 105 Bytnerowicz, A., 41, 50 C Caetono-Anolles, G . , 20, 26 Cakirlar, H., 78, 97, 105 Calba, H., 6, 7, 18, 26 Calheiros, R., 37, 38, 50 Callander, B. A., 47, 50 Cameron, F. K., 127, 182 Cameron, R. S . , 5, 26 Campbell, A. K., 55, 68, 72 Canvin, D. T., 136, 182 Cao, Y.,84, 85, 111 Caporn, S. J. M., 117, 189 Caradus, J. R., 8, 26 Carre, I. A., 68, 72 Carter, C., 135, 136, 140, 148, 167, 188 Cashmore, A. R., 54, 73 Cerana, R., 84, 87, 105 Cerda, A., 120, 127, 177 Chameides, W. L., 47, 49 Chandra, S., 167, 168, 169, 170, 176, 177 Chaney, R. L., 24, 27 Chaney, W. R., 125, 182 Chang, P.-F., 64, 73 Chapin 111, F. S., 19, 26 Chauhan, Y. S., 115, 177 Cheeseman, J. M., 76, 80, 97, 105, 108, 115, 116, 119, 120, 121, 154, 163,

178. 183, 188 Chory, J., 55, 68, 69, 70, 72 Chrerel, I., 84, 106 Christiansen-Weniger, C., 9, 26 Christie, J. M., 54, 55, 59, 60, 65, 70, 71 Chua, N.-H., 54, 55, 61, 62, 63, 68, 69, 70, 72, 73 Claringbold, P. J., 161, 180 Clark, R. A., 128, 158, 159, 161, 176, 177, 185

AUTHOR INDEX

Clarkson, D. T., 5, 26, 123, 147, 178 Cocucci, M., 97, 105 Colavito, L., 41, 51 Collier, G. F., 146, 178 Colling, C., 65, 71 Collins, J. C., 173, 175 Colls, J . J., 34, 49 Colmer. T.D., 77, 108 Colombo, R., 79, 84, 87, 105 Conejero, G., 84, 108 Conti, F., 98, 107 Cory, R., 155, 177 Craig, S., 8, 26 Cramer, G. R., 97, 106, 116, 117, 118, 119, 121, 122, 123, 129, 130, 131, 132, 158, 162, 178, 185 Creelman, R. A., 63, 72 Critchley, C., 167, 187 Cuartero, J., 115, 178 Culligan, K., 68, 72 Curnniing, J. R., 33, 49 Curtis. A. V., 2. 13, 21, 27 Curtis, P. S., 124, 133, 134, 136, 138, 140, 141, 145, 178 Cutt, J. R., 54, 60, 64, 70 Cvitas, T., 38, SO Czempinski, K., 86, 88, 106

D Dahlgren, R. A., 20. 21, 28 Dale, J. E., 125, 178 Daram, P.,84. 106 Davenport, R. J., 100, 106, 120, 178 Davies, M. S., 22, 26, 123. 186 Davies, T. W., 167, 185 Davies, W. J., 118, 164, 179, 189 Dawson, P. J., 41, 50 de Bauer, L. I., 37, 38, 40, 41, 51 De Boer, A. H., 86, 88, 98, 99, 100. 106, 112

De Felice, J., 18, 27 De Silva, D. L. R., 16, 17, 26, 27 De Temmerrnan, L., 32, 35, 50 Delane, R., 115, 117. 118, 119, 121, 125, 134, 137, 140, 179, 185

Delhaize, E., 8, 9, 22. 24, 26. 30 Denby, K. J.. 58, 71 Denecke, I., 60, 61, 73 Deng, X.-W., 55, 69, 70 Derwent, R. G., 46, 50 DeWald, D. B., 63, 72 Dhindsa, R. S., 55, 73

193

Dias, P. L. S., 37, 38, 50 Dietrich, P., 84, 85, 98, 99, 107, 109 Ding, L., 81, 82, 106, 112 Dixon, R. A., 54, 55, 59, 60, 65, 68, 70, 72, 73 Djordevic, M. A., 20, 29 Dobrenz, A. K., 172, 179 Dodd, C., 164, 179 Dodd, W. A., 80, 106 Doering, H.W., 117, 186 Donovan, T., 158, 172, 179 Downton, W. J. S., 115, 117, 119, 120, 130, 131, 132, 158, 179, 187, 188

Dracup, M., 115, 179 Dreyer, I., 84, 85, 98, 105, 107 Dunn, M. A.. 54, 55, 56, 71 DuPont, F. M., 79, 106 Durand, M., 134, 136, 179 Dvorak, J., 119, 120, 187

E Eagle, H. E., 124, 128, 182 Eaton, F. M., 117, 128, 130, 179, 180 Echlin, P., 167, 168, 179, 180 Ecker, J . R., 68, 69, 72 Ehlig, C. F., 128, 176 Ehmann, B., 56, 70, 71 Ehret, D. L., 128, 131, 158, 179 Ehrhardt, T., 86, 88, 106 El-Bahnasaway, R. M., 37.49 Ellenberg, J., 84, 85, 98, 99, 109 Elzam, 0. E., 82, 106, 120. 129, 130. 132. 179

Elzenga, J. T. M., 83, 87, 90, 98, 106 Emmler, K., 56, 71 Enkoji, C., 120, 178 Epstein, E., 82, 97, 106, 115, 120, 129. 130, 131, 132, 158, 162, 172, 178, 179, 182, 185, 187 Erasmus, D. A., 167, 185 Ericson, R. 0.. 164. 187 Esau, K., 125, 147, 179 Eshel, A., 169, 188 Esser, J. E., 99, 108 Ezz El-Din, M. R. M.,37, 38.50

F Fairley-Grenot, K., 98, 99, 106 Falkengren-Grerup, U., 9, 27 Falkenmark, M., 115, 179 Fan, T. W.-M., 77, 108 Fang, H., 115, 186

194

AUTHOR INDEX

Fangmeier, A., 33, 49 Farag, S. A., 37, 49 Favelukes, G., 20, 26 Feldmann, K. A., 68, 69, 72 Felle, H. H., 81, I06 Fernandes, J. C., 162, 179 Fernndez, J. A,, 81, 82, 108 Fernandez, J. M., 83, I10 Fewtrell, C., 169, 177 Findlay, G. P., 78, 84, 86, 87, 89, 90, 91, 92, 95, 97, 98, 99, 100, 103, 106, 108, I l l Fiori, C., 168, 180 Fisher, D. B., 152, 153, 185 Flowers, T. J., 76, 78, 79, 91, 106, 107, 115, 117, 119, 120, 125, 127, 130, 135, 137, 151, 165, 167, 178, 179, 181, 189 Fluhr, R., 54, 71 Fortmeier, R., 130, 134, 138, 140, 141, 145, 179 Fox, D., 86, 109 Foy, C. D., 24, 27 Francis, D., 123, 186 Francois, L. E., 158, 159, 161, 172, 177. I 79 Freijsen, A. H. J., 115, 187 Fricke, W., 117, 118, 164, 165, 179, 180 Frohnmeyer, H., 54, 56, 50, 71 Fuglevand, G., 54, 5 5 , 56, 59, 65, 66, 67, 68, 69, 71 Fuhrer, J., 33, 34, 36, 49, 50, 51 Fujii, T., 79, Ill Fujiyama, H., 128, 181 Fullmer, C. S., 169, 177 Furuya, M., 56, 71 G Caber, R. F., 84, 85, 99, 104, 105, 109, I10 Gajon, A., 24, 30 Galaup, S., 33, 49 Galloway, J. N., 47, 50 Gambale, F., 85, 98, 107 Gandar, G. W., 164, 180 Garbarino, J., 79, 106 Garciadeblas, B., 78, 107 Gardner, P. A., 135, 149, 185 Garrill, A., 84, 86, 87, 89, 90, 91, 92, 95, 98,99, 100, 103, 106, 111 Gassmann, W.,5, 27, 81, 84, 85, 98, 106, 107, 110, I l l Gates, C . T., 159, 161, 180

Gauch, H. G., 117, 130, 180 Gaymard, F., 83, 84, 85, 98, 106, 108, 109, 110, 111 Gebauer, G., 17, 18, 27 Geiger, M.,24, 30 Geissler, P. A., 34, 49 Geletyuk, V. I., 86, 104 Gelli, A., 79, 105 Gibbs, J., 115, 117, 118, 119, 121, 125, 134, 137, 140, 179, 185 Gigon, A., 17, 18, 27 Gilmour, S. J., 55, 71 Gilroy, S., 5 , 6, 28 Gimeno, B.S., 36, 49 Giraudat, J., 55, 68, 71, 72 Glaab, J., 24, 30, 161, 186 Gleick, P. H., 115, 180 Glyer, I. D., 32, 35, 49 Goldsmith, J. G., 168, 169, 170, 180, 183 Goldsmith, M. H. M., 85, 86, 87, 99, 102, 108, 110 Goldstein, J. I., 168, 180 Gonzalez-Fontes,A., 24, 30 Goodman, H. M., 68, 71 Gorham, J., 76, 107. 128, 180 Grabov, A. M., 86,107 Gradmann, D., 80, 83, 85, 86, 87, 97, 98, 100, 102, 104, 105. 107, 109, 110, 111 Graham, I. A., 57, 58, 59, 71 Grandjean, A., 33, 50, 51 Grattan, S. R., 120, 130, 159, 176, 180 Green, P. J., 54, 72 Green, R., 54, 71 Greenway, H., 76, 107, 109, 115, 117, 118, 119, 121, 123, 124, 125, 133, 134, 136, 137, 139, 140, 145, 151, 158, 176, 179, 180, 185 Grieve, C. M., 128, 132, 134, 137, 138, 139, 140, 141, 145, 158, 159, 181, 184 Grignon, C., 84, 108, 110 Grime, J. P., 2, 6, 10, 12, 13, 21, 27, 28 Groneman, A. F,, 9, 26 Grunberg, K., 115, 188 Gug-Ortega, R., 6, 29 Gunn, A., 134, 137, 151, 180 Gustafson, J. P., 8, 26 Gusten, H., 38, 50 H Haarsma, M. S . , 162, 185

195

AUTHOR INDEX

Hahlbrock, K., 56, 59, 60, 65, 71, 72 Hajibagheri, M. A.. 78. 79, 91, 106, 107, 117, 119, 120, 167, 179, 181

Hall, J. L.. 119, 167, 181 Halloran, G. M., 172, 185 Hamill, 0. P.. 82, 107 Handa, A. K., 64, 73 Hansen, U. P., 83, 107 Hanson, J. B., 126, 147, 155. 181, I87 Harmon, A. C., 14, 29 Haro, R., 78, 107 Harris, N., 47, 50 Harter, K., 54, 56, 58, 60. 71 Harter, L. L., 128, 182 Hartung, W., 133, 134, 136, 138, 139, 140, 152, 154, 158, 161, 182

Harvey, B. L., 128, 131, 158, 179 Harvey, D. M. R.. 78, 79, 91, 107, 119, 120, 167, 181

Hasegawa, P. M., 64, 73, 76, 77, 78, 79, 100, 105. 109, 110, 174, I85 Hassan, G . K. Y., 37. 38, 50 Hassan, 1. A,, 40, 42, 50 Hassidim, M., 79, 100, I05 Hauge, B. M., 68, 71 Hauskrecht, M., 133, 176 Havill, D. C., 17, 18, 27 Hawkes, G . R., 128, 181 Hay, R. K. M., 124. 181 Hayat, S., 118, 123. 176 Hayatsu, M., 25, 28 Haydock, K. P., 118, 123, 135, 136, 139,

Hilpert, A., 152, 154, 182 Hiramoto, Y.,101, 109 Hirsch, R. E., 84, 109 Hoagland, D. R., 150, 188 Hodgson, J. G., 6, 12, 27 Hoffen, A,, 169, 188 Hofmann, B., 57, 58, 72 Hope-Simpson, J., 2, 27 Hoshi, T., 98, 99, I07 Hoth, S., 84, 85, 98, 99. 105, 109 Hough, A. M., 46.50 Houghton, J. T., 47, 50 Houlden, G., 46, 49 Houser, J., 115, 186 Hu, H., 163. 181 Hu, Y., 137, 138, 139, 161, 181 Huber, J. J. L., 115, 187 Huffaker, R. C., 160, I82 Hughes, M. A., 54, 55, 56, 71 Hunter, D., 5 , 27 Huntington, V. C., 146, 178 Huprikar, S. S., 84, 104 Hussain, F., 124, 181 I Ichida, A. M., 84, 88, 98, 109 Ilahi, I., 124, 181 Ilyas, M., 115, 171, 172, 186 Imamul Huq, S. M., 118, 181 Itai, C., 126, 176 Iwasaki, N., 101, I09 Izard, P., 125. 135, 189

140, 141, 145, 159, 161, 180. 189

Heck, W. W., 32, 35, 50 Hedrich, R., 82, 83. 84, 85, 98, 99, 105, 107, 109, 110

Heime, R., I I , 27 Heino, P.,55, 73 Heinrich, G., 37, 38, 50 Heller, R., 16, 28 Hemmingsen, S., 78, 107 Henriques, F. S., 162, 179 Hepler, P. K.. 17, 27 Hermondson, M. A., 64,73 Hess, F. D., 78, 79, 105 Hether, N. H., 12, 27 Hetherington, A. M., 14, 17, 26. 27, 30 Hewitt, E. J., 4, 27 Higashi, R. M., 77, 108 Hill, J., 155, 181 Hille, B.. 82, 83, 107 Hillniann, T., 168, 184

J Jabs, T., 65, 71 Jackson, I. L., 12, 27 Jackson, J. A., 55. 56, 65, 66, 67, 68, 69, 71

Jacoby, B., 79, 100, 107. 109. 112 JaffrC, T., 11, 27 Jagadish, V. C., 8, 26 Jager, H. J., 32, 34, 35, 50, 51 Jaillard, B., 6, 7, 18, 26 Jang, J.-C., 58, 71 Jefferies, R . L., 16, 27 Jeftic, J., 38, 50 Jenkins, G. I., 54, 55, 56, 5 8 , 59, 60, 65, 66, 67, 68, 69, 70, 71, 73

Jeschke, W. D.,, 117, 119, 124, 133, 134, 135, 136, 137, 138. 139, 140, 141, 152, 153, 154, 155, 157, 158. 159, 161, 176, 181, 182, 186. 189

196

AUTHOR INDEX

Jia, Z.-P., 78, 107 Johannes, E., 83, 107 Johnson, C.M., 9,26 Johnson, J., 6, 29 Johnson, P. A., 6, 27 Jones, D. L., 5, 6, 27, 28 Jones, R. L., 85, 105 Joy, D. C., 168, 180 Jurgens, G., 69, 72

K Kahane, I., 126, 182 Kaiser, T., 56, 71 Kaiser, W. M., 91, I l l , 117, 160, 161, 182, 186, 187, 188 Kakutani, T., 86, 88, 103, 109 Karlsson, P. E., 34, 51 Kasamo, K., 79, 111 Kasana, M. S., 44, 45,50 Kashibhatia, P.S., 47, 49, 50 Kats, G., 41, 50 Katsuhara, M., 78, 97, 99, 107, 108 Kattenberg, A., 47, 50 Kawasaki, T., 130, 131, 182 Kay, S. A., 68, 72 Kearney, T. H., 127. 128, I82 Keiber, J. J., 55, 68, 69, 72 Kelly, W. B., 99, 108 Kernp, D. R., 124, 181 Kent, B., 119, 167, 181 Kent, L. M., 129, 182 Kern, R., 61, 73 Kielland, K., 19, 26 Kikuyama, M.,101, 109 King, P., 41, 51 Kingsbury, R. W., 115, 172, 182 Kinraide, T. B., 5, 8, 28, 30 Kinzel, H., 16, 28 Kircher, S.,54, 60, 71 Kirchhoff, V. W. J. H., 37, 38, 50 Kirst, G. 0.. 76, I09 Kjellbom, I?, 65, 70 Klagges, S., 152, 154, 182 Klasine, L., 38, 50 Klessig, D. F., 54, 60, 64, 70 Klobus, G., 160, 182 Knight, H., 5 5 , 72, 101, 108 Knight, M. R., 55, 68, 72, 101, I08 Kobayashi, K., 41, 50 Koch, K. E., 57, 59, 72 Kochian, L. V., 5, 6, 7, 8, 27, 28, 29, 84, 104, 162, 182

Kohut, R. J., 41, 51 Kosuge, N., 25.28 Kourie, J. I., 78, 85, 97, 99, 108 Kramer, P. J., 118, 182 Krapp, A., 57, 58, 72 Krenz, M., 54, 60, 71 Kretsch, T., 56, 71 Kristiansen, L., 34, 49 Krysan, P. J., 82, 108 Kubica, S., 133, 176 Kuch, A., 98, 107 Kuhn, W., 146, 150, 156, 188 Kuiper, P.J. C., 120, 127, 132, 184 Kumar, R., 82, 110 Kuznetsov, V. V., 174, 182 Kylin, A., 120, 127, 132, 184 1 Lacan, D., 134, 136, 179 Lacroute, E, 84, I10 Lado, P., 79, 105 Lagarde, D., 84, 108 Lagares, A,, 20, 26 Lagerwerff, J. V., 124, 128, 182 Laguette Rey, H. D., 40, 41, 51 Lam, E., 54, 72 Lamart, A., 16.28 Lamb, C.J., 65, 70, 72, 73 Lamoreaux, R. J., 125, 182 Larher, E, 118, 181 Lass, B., 81, 108 Liiuchli, A., 77, 96, 97, 101, 106, 108, 110, 119, 120, 123, 124, 125, 127, 129, 130, 132, 133, 134, 135, 136, 137-8, 139, 140, 141-2, 143, 144, 145, 146, 147, 148, 149, 150, 152, 154, 155, 156, 157, 158, 159, 162, 163, 165, 167, 168, 177, 178, 182, 183, 184, 188 Lauer, M., 24, 30 Laurence, J. A., 41, 51 Laurie, S., 83, 84, 86, 87, 88, 91, 92, 95, 97,98, 99, 102, 103, 104 Laycock, D., 16, 27 Layzell, D.B., 136, 182 Lazaroff, N., 131, 158, 183 Lazof, D., 80, 108 Lazof, D. B., 120, 121, 124, 125, 134, 136, 138, 141-2, 143, 144, 145, 146, 147, 148, 149, 154, 158, 161, 167, 168, 169, 170, 171, I83 Le Gales, Y., 16, 28

AUTHOR INDEX

Lea-Cox, J. D., 161, 183 Leaver, C. J., 58, 71 Lechno, S., 117, 183 Lee, J. A., 11, 13, 17, 18, 21, 22, 24, 27, 28 Lee, K. S., 125. 135, 189 Lee, R. B., 25, 28 Lehman, H., 167, 188 Lehnen, M., 84, 105 Leidi, E. 0.. 120, 183 Leigh, R., 83, 84, 86, 87, 88, 89, 90. 91, 92, 95, 97, 98, 99, 100, 102, 103, 104, I l l . 117, 164, 165, 180, 184 Lemaillet, G., 85, 98, I09 Lemtiri-Chlieh, F., 87, 90, 92, 98, 100, 112 Lenherr, B., 33, 51 Leon, P., 58, 71 Lepetit, M., 84, 108 Lerner, H. R., 79, 100, 105 Lessani, H., 120, 130, 131, 152, 184 Leung, J., 68, 72 Levine, A,, 65, 72 Levy 11, H., 47, 49, 50 Li. H.-M., 68, 72 Li, W., 99, 108 Li, W. W., 99, 108 Libbenga, K. R., 86, I l l Lifshin, E., 168, I80 Ling, Y. C., 170, 176 Linton, R. W., 149, 168, 169, 170, 171, 180, I83 Liu, D., 64, 73 Liu, J., 118, 184 Logemann, E., 59, 71 Lois, R., 60, 72 Long, J. C.. 55, 71 Long, M. J., 124, 135, 187 Lowendorf, H. S., 20, 28 Lozoya, E., 60, 72 Luan, S., 99, 108 Lucas, W. J., 84, 85. 104, 110 Luckhardt, R. L., 128, 181 Liittge, U., 80, 108, 170-1, 184 Lynch, J.. 97, 101, 106, 108, 123, 129, 132, 134, 137, 138, 141, 145, 158, 184

M Maas, E. V., 120, 128, 130, 132, 134, 137, 138, 139, 140, 141, 145, 158, 159, 180. 181. 184 Maathuis, F. J. M., 81, 82, 84, 86, 87, 88. 90. 91, 92, 98, 108, 109. I12

197

Macduff, J. H., 160, I86 Machida, Y., 58, 73 Machler, F., 33, 51 Mackay, A. D., 8, 26 MacKenzie, A. J., 119, 128, 175 Maggs, R., 39, 40, 41, 43, 44, 45, 51. 52 Mahmoud, A., 10, 28 Maksymowych, R., 125, 184 Malone, M., 164, 184 Mansfield, P. J., 46, 49 Mansfield, T. A,, 16, 17, 22, 26, 27, 29 Mariano, M. M.. 37, 38, 50 Marinho. E. V. A., 37, 38, 50 Marrt., E., 97, I05 Marschner, H., 12, 14, 28, 29, 77, 79, 109, 120, 127, 130, 131, 132, 152, 162, 184, I89 Marsh, E. L., 82. 110 Martel, R., 78, 107 Marten, I., 85.98, 109 Martin, P., 156, 184 Martinez, V., 120, 127, 134, 139, 140, 149, 154, 155, 156, 157, 158, 159. 177, 184

Marty, A., 82, 107 Maruyama, S., 147, 184 Maskall, K 47, 50 Made, J., 164, 176 Mason, H. S . , 63, 72 Mass, E. V., 158, 172, 179 Mathy, P., 32, 35, 50 Matsuda, K., 121, 126, 184, 187 Matyssek, R., 147, 184 McAinsh, M. R., 14, 28, 30 McCarl, B. A,, 32, 35, 49 McCullough, N., 78, 107 McGrath, R. B., 55, 61, 72 McIntye, G . I., 161, 184 McKendree, W. L., 84, 109 McNeill, S., 46, 49 McNeilly. T., 115. 120, 129, 130, 131. 158, 171, 172, 173, 175, 176 McVickar, M. H., 128, 181 Meira Filho, L. G., 47, 50 Meleigy, M. I., 37, 49 Mendoza, N. M., 40,41, 51 Mennen, H., 79, 109 Meriot, S., 55, 68, 72 Mesnick, L., 115, 186 Meyer, R. F., 118, I84 Michaels, A., 47, 50 Michel, M.,168, 184

AUTHOR INDEX

Michelina, V. A., 118, 184 Millar, A. J., 55, 61, 68, 72 Millard, P. J., 169, 177 Miller, P., 37, 38, 51 Millhouse, J. A.. 117, 119, 187 Millikan, C. R., 155, 156, 184 Mimura, T., 99, 108 Minet, M., 84, I10 MisBra, S., 69, 72 Mohr, H., 63, 73 Mol, J., 55, 59, 60,73 Mol, J. N. M., 59, 60, 73 Mollanen, L., 19, 26 Monnich, E., 37, 38, 50 Monroy, A. F., 55, 73 Moran, N., 86, 109 Moran, O., 98, 107 Morcuende, R., 24, 30 Morecroft, M. D., 17, 18, 28 Morgan, A. J., 167, 184, 185 Morgan, S. M., 18, 28 Modtsugu, M., 130, 131, 182 Morrison, G. H., 167, 168, 169, 170, 176, 177, 186 Miiller, A. J., 69, 72 Muller-Rober, B., 84, 85, 86, 88, 98, 99, 106. 109 Mullet, J. E., 63, 72 Mummert, H.,80,109 Munns, R.. 76, 77, 107. 109. 115, 117, 118, 119, 121, 123, 124, 125, 126, 135, 137, 146, 149, 152, 153, 157, 158, 174, 176, 180, 185, 187, 188, 189 Munns, S.,118, 121, 134, 140, 179 Muranaka, T., 58, 73 Murata, Y., 86, 88, 103, I09

Neuwinger, K., 98, 107 Neves Piastun 165 Newbury, D. E., 168, 180 Nieman, R. H., 158,185 Niu, X., 76, 77, 109, 174, 185 Noble, C. L., 172, 185 Noguchi, M., 86, 88, 103, 109 Nolasco, A. Q., 37, 38, 51 Nomoto, K., 12, 30 Nonami, H., 147, 185 Nordin, K., 55, 73 Norlyn, J. D., 172, 179 Northup, R. R., 20, 21, 28 Norton, R. A., 155, 185 Novacky, A., 81, 111 Novak, R. S., 162, 178 Nurnburger, T., 59, 71

N Nagata, T., 25, 28 Nagatani, A., 56, 71 Nagy, E, 54, 60, 71 Nakajima, H., 65, 73 Nakamura, R. L., 84, 109 Nakamura, Y., 37, 38, 50 Nakayama, F. S., 155, 177 Nandi, P. K., 44.45, 49 Narasimhan, M. L., 64,73 Neher, E., 82, 84, 85, 86, 107, 110 Nelson, C. J., 164, 187. 188 Neuhaus, G., 61, 62, 63, 68, 70, 73 Neumann, I?, 118, 185 Neumann, P. M., 100, 112

P Paiva, L. A., 54, 55, 59, 60, 70 Palme, K., 84, 98, 105. 107 Palmer-Brown, D., 36, 49 Palva, E. T., 55, 73 Palva, T. E., 60, 61, 73 Palzkill, D. A., 150, 156, 186 Papernik, L. A., 8, 29 Papp, J. C., 124, 186 Parcy, F., 68, 71 Pardines, J., 120, 127, 177 Pardo, 5. M., 76.77, 109, 174, 185 Parker, D. R., 5, 28 Parniske, M., 59, 71 Passioura, J. B., 118, 188

0 Obermeyer, G.,87, I09 Obi, I., 86, 88, 103, 109 Oertli, J. J., 117, 185 Okazaki, Y.,101, 109 O’Leary, J. W., 100, 104 Olsen, C., 16, 29 Olsen, R. A., 12, 27 Olson, R. K., 20, 26 Olsson 13, 25, 30 Olszyk. D., 41, 50 Olszyk, D. M., 35, 51 Omelian, 3. A., 131, 132, 158, 185 Osmond, C. B., 80, 108, 119, 180 Otte, M. L., 162, 185 Ourry, A., 160, 186 Ownby, J., 6, 29

199

AUTHOR INDEX

Pate, J. S., 133, 136, 152, 153, 155, 156, 157, 158, 159, 161, 181, 182, 186 Pei, Z.-M., 88, 112 Penkett, S. A., 46, 51 Pereira, E. B., 37, 38, 50 Perez-Alfocea, F.,160, I77 Peuke, A. D., 161, 186 Pfankoch, E., 64, 73 Pick, U., 79, 109 Pigott, C. D., 2, 29 Pimentel, D., 115, 186 Pineros, M., 5 , 29 Piston, D. W., 170, 187 Pitman, M. G., 80, 106, 128, 131, 134, 135, 136, 137, 140, 148, 151, 158, 167, 183, 186, I88 Pleijel, H., 34, 41, 51 Poe, M. P., 35.51 Poethig, R. S., 125, 186 Polito, V. S., 101, 108 Poljakoff-Mayber, A., 126, 182 Pollard, A., 77, 109, 112 Ponce de Le6n, I., 60, 61, 73 Powell, M. J., 123, 186 Preiss, E., 115, 186 Prins, H. B. A., 88, 111 Provart, N., 84, 85, 98, 99, 109 Prudhomme, M. P., 160, 186 Puthota, V., 6, 29

Q

Quatrano. R. S., 63, 70 Qureshi, R. H., 115, 171, 172, 186 Ragjpthama, K. G., 64,73 Rains, D. W., 76, 82, 106, 109 Rajasekhar, V. K., 65, 73 Ramadan, A. B., 37, 38, 50 Ramseyer, G. O., 170, 186 Rana, R. S., 172, 186 Randall, P. J., 8, 9, 22, 26, 30 Rao, D. N., 44, 45, 49, 51 Raschke, K., 84, 85. 86, 87, 89, 98, 99, 100, 110, 112 Ratcliffe, R. G . . 25, 28 Rathert, G., 1 1 7, 186 Rawer, W. E., 126, I87 Raven, J. A., 22, 29 Ravina, I., 100, 112 Rawson, H. M., 121, 124, 126, 135, 149, 185, 187 Read, D. J., 20, 29

Read, N. D., 55, 73 Redmann, R. E.,,128, 131, 158, 179 Regnier, F. E., 64, 73 Rehder, H., 17, 18, 27 Reid, D. A., 8, 29 Reid, J. D., 84, 85, 99, 105 Reid, R. J., 81, 100, 106, 111, 120, 178 Reimann, C., 120, 187 Reiners, W. A., 20, 26 Reinhold, L., 79, 100, 105 Reinold, S., 59, 71 Rengel, Z., 97, 109, 118, 187 Reuveni, M., 100, 110 Rhodes, P. R., 126, 187 Riazi, A., 121, 184 Richardson, A. E., 20, 29 Ridout, M., 83, 87, 89, 112 Riego, D. C., 6, 8, 26 Ringoet, A,, 155, 187 Ritchie, G. S. P.,5, 26, 29 Rizk, H. F. S., 37, 49 Roadknight, C., 36, 49 Roberts, D. M., 14,29 Roberts, S. K., 85, 86, 87, 89, 90, 91, 92, 94, 95, 98, 99, 100, 110 Robins, M. F., 118, 123, 135, 136, 139, 140, 141, 145, 159, 161, 180, 189 Robinson, D., 16, 29 Robinson, S. P., 117. 119, 187 Robson, A. D., 5 , 26 Rocholl, M., 56, 71 Rodriguez-Navarro. A,, 78, 107 Rolfe, B. G., 20, 29 Roman, G., 68, 69, 72 Romheld, V., 12, 29. 162, 189 Rook, F., 57, 59, 73 Rorison, I. H., 2, 5, 16, 17, 18, 27, 29 Ross, D. S., 5, 27 Rothenberg, M., 68, 69, 72 Rozema, J., 115, 162, 185, 187 Rozema-Dijst, E., 115, 187 Rubio, E, 81, 107, 110 Rufty, T. W., 161, 168, 169, 183 Ruiz, L. P., 16, 17, 22, 26, 29 Runge, M., 17, 29 Rush, D. W., 120, 172, 187 Rusnak, F., 99, 108 Ryan, P. C., 8, 24, 26 Ryan, P. R., 5, 8, 9, 22, 26, 28, 30 S

Sachs, T., 125, 187

200

AUTHOR INDEX

Sacks, W. R., 59, 71 Sairz, J. F., 120, 183 Sakman, B., 82, 107 Salmon, J. M., 84, 110 Sanders, D., 79, 81, 82, 83, 84, 86, 87, 88, 90, 91, 92, 95, 97, 98, 99, 102, 103, 104. 107, 108, 109, 111. 112 Sanders, G. E., 34, 51 Sanders-Mills, G., 36, 49 Sandison, D. R., 169, 170, 177, 187 Sarwar, G., 152, 154, 182 Satoh, S., 79, 111 Satter, R. L., 86, 109 Sauer, G., 155, 187 Scacchi, A., 97, 105 Schachtman, D.P., 82, 85, 86, 88, 103, 110, 119, 120, 187 Schafer, C., 57, 58, 72 Schlfer, E., 54, 55, 56, 58, 59, 60, 70, 71, 73 Schatzler, H. P., 146, 150, 156, 188 Schauf, C. L., 83, 86, 90, 110 Scheel, D., 59, 60, 65, 71, 72 Scheible, W.-R., 24, 30 Schlesinger, W. H., 47, 50 Schmelzer, E., 59, 71 Schmidhalter, U., 137, 138, 139, 161, 181 Schmidt, C., 116, 117, 118, 121, 122. 116, 130, 132, 178 Schmidt, R., 58, 73 Schnoor, J. L., 47, 50 Schnyder, H., 164, 187 Scholz, G., 156, 188 Schonhurst, M. H., 172, 179 Schreck, J., 115, 186 Schreiber. S. L., 99, 108 Schroeder, J. I., 5, 27, 81, 82, 83, 84, 85, 86, 88, 98, 99, 105, 106, 107, 108, 109, 110, 111, 112 Schubert, S., 77,96, 110 Schulze, E.-D., 24, 30 Schumaker, S., 100, 104 Sedbrook, J. C., 84, 109 Seemann, J. R., 167, 187 Sillden, G., 34, 41, 51 Sellers, E. K., 17, 18, 28 Sentenac, H., 83, 84, 85, 98, 99, 105, 106, 109, 110, 111 Serrano, R., 76, 77, 110 Setter, T. L., 117, 124, 133, 134, 136, 139, 140, 158, 176 Shacklock, P. S., 55, 73

Shah, D., 34.49 Shamsi, S. R. A., 39, 40, 42, 43, 51, 52 Shannon, M. C., 128, 159, 187 Sharkey, P. J., 155, 186 Shaw, E. J., 128, 181 Shaw, M.J., 56, 68, 69, 71 Sheen, J., 57, 73 Sheen, J.-C., 58, 71 Shevyakova, N. I., 174, 182 Shibata, J. K., 40, 41, 51 Shirasu, K., 65, 73 Shirzadegan, M., 174, 189 Sigworth, F. J., 82, I07 Silk, W.K.,124, 137, 138, 139, 140, 145, 147, 150, 158, 163, 164, 165, 177, I87 Simpson, R. J., 20, 29 Sims, A. P., 16, 27 Singh, M., 44, 45, 51 Singh, N. K., 64, 73 Skarby, L., 34, 36, 41, 49, 51 Skerrett, I. M., 100, 111 Skerrett, M., 84, 87, 89, 90, 91, 92, 95, 98, 99, 100, 103, 106, Ill Slayman, C. L., 85, 86, 87, 99. 102, 105, 110

Smalle, J., 68, 71 Smeekens, S., 57, 59, 73 Smith, C. A.. 169,177 Smith, D., 172, 179 Smith, E A., 22, 29, 81, 82, 100, 106, 108, 110. 111, 120, 178 Smith, J. A. C., 79, 104 Smith, S. M., 55, 68, 72 Snaydon, R. W., 5, 22, 26 Snowden, R. E. D.,10, 30 Sod, E. W., 168, 177 Sonnewald, U., 58, 73 Spalding, E. P., 86, 87, 102, 110 Speer, M., 91, 111, 160, 187, 188 Sprung, D.,37, 38, 50 Spurr, A. R., 129, 130, 131, 158, 178 Stapleton, A. E., 54, 59, 73 Stein W. D., 80, Ill Steizer, R., 120, 130, 135, 136, 140, 148, 167, 168, 188

Steveninck, M. E., 120, 130, 188 Steveninck, R. F. M., 135, 139, 159, 188 Stevens, C. S., 37. 38, 51 Stewart, G. R., 16, 17, 18, 21, 24, 27, 28 Stitt, M., 24, 30, 57, 58, 72, 73 Stoeckel, H., 87, 111

20 1

AUTHOR INDEX

Stolo, A. L. H., 168. 177 Stone, J. E.. 172, 179 Storey, R., 135, 136. 140, 148, 167, 188 Stout, P. R., 150, 188 Strayer, C. A., 68, 72 Strijm, L.,, 14, 15, 22, 30 Stuitje, A. R., 59, 60, 73 Stumm, W., 5 , 30 Suggs, C., 168, 169, 183 Sunderland. N.. 125. I88 Sussex, 1. M., 125, I86 Sussnian, M. R., 82, 84, 108, 109 Szabolcs, I., 76, 111

Tonneijck, A. E. G., 32, 36, 51 Tonnet, M. L.. 135. 149, 152, 153, 157, 158, 185, 189 Toroksalvy, E., 117, 120, 188 Treeby, M. T., 135, 139, 159, IN8 Trewavas, A. J., 5 5 , 68, 72, 73, 101, 108 Troke, P. F., 76, 106, 119, 120, 127, 179 Tschope, M., 65, 71 Tyemian, S. D., 84, 86, 87, 88, 89, 90, 91. 92, 95, 98, 99. 100, 103, 106. 110. 111 Tyler, G., 9, 13, 14, 15, 22, 25, 27, 30

U T Takagi, S., 12, 30 Takeda, K., 87, I l l Takemoro, T., 12, 30 Taleisnik, E., I 15, 188 Take-Messerer, C., 58, 71 Tansley. A . G., 2, 30 Tariche, S., 115, 186 Tawfik, F. S., 38, 50 Tax, E, 82, I08 Taylor, G., 6, 7, 30 Taylor, J. E., 14, 30 Taylor, 0. C., 32. 35, 50 Taylor, S. E., 167, 168. 179 Tazawa, M., 78, 97, 99, 107, I O N Tejeda. T. H., 37, 38, 51 Tel-Or, E., 117, 183 Tenhaken, R., 65, 72 Teomy, S., 79, 107 Termaat, A., 115. 118, 125, 146, 185, 188 Terry, B. R., 86, 88, 103, 110, If1 Terry, N., 119, 124, 176, 186 Terzaghi. W. B., 54. 73 Tester, M. A,, 5, 29, 82, 85, 86, 87, 89, 90, 91, 92, 94, 95, 98, 99, 100, 110, 111, 112 Thibaud, J.-B., 83, 84, 85, 98, 109. I l l Thiel, G . , 98, 99, 100, 102, I l l , 123, 134, 137, 138. 141, 145, 158. 184 Thomas, D. A., 134, 137, 151, 180 Thomashow, M. F., 55, 71 Thompson, C . R.. 35, 41, 50, 51 Thompson, R. K., 172, 179 Tibbitts, T. W., 150. 156, 186. 188 Tingey, D. T.. 32, 35, 50 Tomos, A., 164, 184 Tomos, A. D.. 117, 118. 165, 175, I80 Tomos, D., 164

Udo, W. S., 156. 188 UK FQRG 32, 51 Ullrich-Eberius, C. I.. 81, 108, Ilf Unsworth, M., 32, 35. SO Uozumi, N., 84, 85, 111 Urbach, S., 84, 106 Urwin, N. A R., 58, 59, 73

V Vaddia, Y.,126, I76 Valon, C., 68. 71 van der Eerden, L. J.. 32. 36. 51 van der Meer, I. M., 59, 60, 73 Van Duijn, B , 86, 111 van Leuken, P., 41, 51 van Steveninck, R. F. M., 120, 130, 188 van Veen, J. A,, 9, 26 Van Volkenburgh, E., 83, 87, 90, 98, 106. 118, 188 Vanbel, A. J. E., 81, 111 Venables, A. V., 115, 188 Verlin, D., 81, 82, I O N VCry, A. A.. 83, 84, 85, 98, 111 Vidal, S., 60, 61, 73 Vogelzang, S. A,, 88, 111 Vogt, K. A,, 20, 21, 28 Volenec, J. J., 164, 188 Volk, R. J., 149, 170, 171, 183 Volpe, C., 37, 38, 50 von Schaewen, A., 58, 73 von Tiedemann, A., 34, 51

W Wada, M., 79. 111 Wahid, A., 39, 40, 42, 43, 51, 52 Waisel, Y., 169, 188 Walker, N. A,, 81, 82, 108. 110, 111, 112 Walker, R. R., 117. 120, 188

202

AUTHOR INDEX

Wallihan, E. F., 12, 30 Wallin, G., 34, 41, 51 Ward, J. M,, 88, 98, 110, 112 Ward, M. R., 160, 182 Wasserman, R. H., 169, 177 Watkin, E., 117, 124, 133, 134, 136, 139, 140, 158, 176 Wayne, R., 0. 17, 27 Webb, A. A. R. 14.30 Webb, W. W. 169, 170, 177, 187 Wegner, L. H. 86, 87, 88, 89, 98, 99, 100, 106, 112 Weigel, H. J. 34, 49, 51 Weigl, J. 170-1, 184 Weiland-Heifecker, U. 69, 72 Weiss, D. 55, 59, 60, 73 Weisshaar, B. 56, 71 Weppner, J. 37, 38, 50 West, D. W. 172, 185 West, K. R. 80, 106 Wheeler, B. D. 10, 30 White, M.C . 24, 27 White, 0. 115, 186 White, P. J. 82, 83, 87, 89, 90, 92, 93, 94, 98, 100, 112 WHOLJNEP 36,52 Wickens, L. 116, 188 Wickens, L. K. 116, 120, 178 Wiebe, H. J. 146, 150, 156, 188 Wieland, E. 5, 30 Wienecke, J. 119, 120, 135, 136, 137-8, 141, 144, 145, 146, 152, 156, 182, 188

Wijnands, J. H. M. 32, 36, 51 Wilkins, D. A. 115, 188 Willis, A. J. 16, 27 Willmitzer, L. 84, 85, 98, 99, 109 Wilmitzer, L. 58, 73 Wilson, J. R. 118, 123, 124, 135, 136, 139, 140, 141, 145, 159, 161, 189 Wilson, K. J. 83, 86, 90, I10 Winicov, I. 174, 189 Witt, J. 117, 186 Wittwer, S. H. 155, 162, 177

Wolf, J. W. 41, 50 Wolf, 0. 124, 133, 134, 135, 136. 137, 138, 139, 140, 141, 152, 153, 154, 157, 158, 159, 161, 181, 182, 189 Wollenweber, B. 17, 18, 27 Wong, M. 54, 72 Woolhouse, H. W. 7, I I , 13, 22, 28, 30 Wu, L. 119, 176 WU, S.-J. 81, 112 WU,W.-H. 99, 102, 112 Wu, Y. 147, 185 Wyn Jones, R. G. 77, 89, 104, 109, 112

x Xu, Y. 64, 73 Y Yamagata, H. 61, 62, 63, 68, 70 Yeo, A. R. 76,91,106, 115, 117, 119, 120, 125, 127, 130, 135, 137, 151, 152, 156, 163, 165, 167, 178, 179, 181. 189 Yermiyahu, U. 8 , 3 0 Yienger, J. 47, 49 Yoshihashi, M. 86, 88, 103, 109 Young, J. C . 82, 108 Young, P. G. 78,107 Ypey, D. L. 86, 111

2 Zambrzuski, S. I1 9, 176 Zamski, E. 117, 183 Zayed, A. 119, 176 Zengshou, Y. 20, 21, 28 Zhang, C. 162, 189 Zhang, J. 118, 189 Zhou, L. 58, 71 Zhu, J. K. 118, 184 Zhu, J.-K. 81, 82, 106, 112 Zidan, I. 100, 112 Zierold. K. 167, 189 Zimmermann, S. 86, 88, 106 Zingarelli, L. 79, 104

SUBJECT INDEX

A abscisic acid-dependenthndependent pathway 35 abscisic acid signal transduction 68 Acetubuluriu 80, 97, 101 Agmpyron 132 Agropyoti elongututii 159 Agropvroti intermecliuni 129, 130 Agrosris cupilluris 7 aluminium and acidity in soil, 4-10 effects on plasma membrane and cytosolic processes 2 2 4 stimulation of malate synthesis 22. 24 aluminium tolerance 610. 22 aluminium toxicity 5-6, 18-20 Aly.viu rubricucrlis 1 I ammonium assimilation 22 extractable, in soil 2, 4 Anthoxunthuni odorarum 5 , 22 Arubidopsis 56, 65, 67, 68, 8 I , 84, 100, 102 Arrhenutherum elutius 10, 11 atmospheric vapour pressure, ozone uptake and 33 ATP, cation influx across plasma membrane and 102 Atrip1e.x 79, 80, 144 Arriplex uninicolu 124, 136, 139, I40 Atriplex spotigiosu 136, 140, 148 Avenu IS5 Azospirillum 9

B Befa wlguris 124, 132 beta-glucuronidase(GUS) reporter gene 61 bicarbonate in soil 4, 22 toxicity 11-13

BIOLOG plate technique 25 blue light signal transduction pathway 65-7 boron in soil 4, 20-1 Brrrssicu F1trpU.S 58 Brussicu olerucea I59 Bmniopsis heneketiii 9 BroninpsiA erecfu 18 Bronius erectci

C CAB genes 5 8 , genes 61-3 C&nus cujun 45 calcium disturbed, in young tissues 137-8 effects on plasma membrane and cytosolic processes 2 2 4 influx across plasma membrane 97-1 01 in interactions between signalling pathways 68 recirculation in the shoot 155-6 salinization, transport and 157-8 in soil 2, 4, 14-17 translocation to the shoot 129-3 I transport in growing shoot tissues 144 calcium ion permeable channels 5 Crirnelliu sinensis 8 Cupsicum antiuuni 130, 152 Carex pilulijeru 13 Curthumnus tinctorius 152 cation influx across plasma membrane 96- 103 ATP 102 cytosolic calcium and pH 101 external and cytosolic sodium 101-2 external calcium and pH 97-1 0 I voltage 97 Centururea scubinsu I6 Chuetnmorphu 80 chalcone synthase (CHS) genes 55, 56, 60 Charu 81, 101

204

SUBJECT lNDEX

Chara injlata 97 Chenopodium 57, 58 chlorosis, lime-induced 12 CHS promoter activity 61-3 Cicer arietinum 45 Citrus 161 Citrus reticulata 117 cobalt in soil 4, 20 Commelina communis 16 compartmental analysis by efflux 154-5, 163 Crassulacean Acid Metabolism (CAM) 22

D Ductylis glomerata 130, 131 Daucus carota 159 defence genes, regulation of 64-5 defence responses 59-61 Deschampsia flexuosa 10, 11, 18 dose-response relationship 56

E EDU 34,41-2 electron probe X-ray microanalysis (EPXMA) 163, 167-8 elemental deposition rates 163-6 Elodea 81 EPXMA (electron probe X-ray microanalysis) 163, 167-8 Ericaceae 18, 24 Erwinia carotovora 60-1 ethylene signal transduction 68 European Crop Loss Assessment Network 35-6 exposure-response studies to ozone 34-6

F Fesruca rubra 130, 131 FHC (frozen hydrated cryofractured) EPXMA 167-8 flavonoid biosynthetic pathway 59-61 fluorescence 170 frozen hydrated cryofractured (FHC) EPXMA 167-8

G Galium saxatile 2, 13 Galium sterner; 2 gene expression responses 54-5, 57-9 control by metabolites 57-9 genotypic sensitivity to salinization 131-2 Glycine 139, 140, 161

Gl.ycine max 42, 120, 126, 136, 137, 14I , 152, 159 Glycine tomenrella 136, I59 Glycine wightii 136, 161 Goldman-Hodgkin-Katz (GHK) equation 83, 92 Gossypium hirsutum 129, 139, 140, 159 growth dilution 164 growth kinetics analysis (GKA) 163-6 Glycine wightii 159

H Helianthus annuus 126 hexokinase, sugar signalling pathway and 58-9 Hibiscus 144, 145 Hibiscus cannabinus 45, 124, 136, 138, 140, 141 Holcus lanatus 18, 130, 131 Hordeurn 121, 144, 145 Hordeum vulgare 123, 124, 125, 129, 130, 131, 132, 137, 138, 141, 152, 153, 156, 161, I62 hypersensitive response (HR) 65

I inward-rectifying channels (IRCs) 84-8 ion excess hypothesis 119, 133, 173-4 iron acquisition 22 extractable, in soil 4 iron deficiency 11-13 iron toxicity 10-11 jasmonic acid (JA) 55 Juncus squarmsus 16

J

K Kerr Gapon equation 128 kinematic growth analysis 163-6 Koeleria mncranthu 11

L Luctuca 144, 145 Luctuca saliva 120, 124, 125, 129, 130, 131, 136, 139, 140, 141-3, 154, 159 Lens culinaris 45, 1 18 Leantodon hispidus 16 Lepidium sativunz 130 Leptochloa fusca 152, 154 lime-induced chlorosis 12

205

SUBJECT INDEX

linear variable differential transformers (LVDTs) 116 Lolium perennr 130, 13 I long-term recirculation of nutrients 151 4 Lophopyrum cdonguturn 1 3 1 , 132 Lupinus 144, 145 Lupinus ulbus 131, 136, 138, 139, 140, 152, 153, 154, 156, 157, 161 Lupinus Iicteus 16, 130, 139, 159

M magnesium extractable, in soil 2 salinization and transport of 13940, 158 translocation to the shoot 130-1 transport in growing shoot tissues 144 malate, effect of aluminium on 33 malatekitrate aluminium detoxification mechanisms 25 manganese toxicity 1&11 Maytenus biireaviann 11 Mrdicago sativu 45. 129, 17 1 Mesetnbrycmthernurn ctystallinurn 79 methyl jasmonate (MeJA) signalling pathway 63, 64 microautoradiography 170-1 microdissection 166 microelements salinization, transport and 162 in soil 20-1 molybdenum deficiency 2&1

N National Crop Loss Assessment Network (NCLAN) (US) 34-5 Nicotiancr tahacum 126 Nitrlla 81 Nitrllopsis 97, 101 nitrate assimilation 22 in soil 4 utilization 17-18 nitric oxide (NO) 42 nitrogen salinization, transport and 160-3 in soil 17-20 nitrogen dioxide as air pollutant 39, 41. 45, 46 nitrogen oxide emissions 32, 46, 47-8 nutrient status and transport, microscale Study 162-71

0 Origanirm vitlgare I6 Otyza 144 Uryza sativa 117, 124, 130, 151 osmoregulatoryhrgor hypothesis of salt tolerance 1 17-1 8, 173 Osrnotin 64 outward-rectifying channels (ORCs) 88 ozone adverse effects on crops 3 9 4 2 exposure-response studies 34-6 field chamber studies 34, 3 9 4 1 formation reactions 32 future concentrations 46-7 impacts on agricultural crops 33-6 ozone protection chemicals studies 34, 41-2 response of tropical crops and cultivars 43-6 experimental studies 43-5 field studies 45-6 rural levels in developing countries 36-9

P patch clamp technique 24-5 pathogenesis-related (PR) proteins 60 Persea americana 130, 13I , 132 Phaseofus I55 Phaseolus vulgaris 7, 36. 42, 58, 124, 125, 130, 131, 151 phenylalanine ammonia-lyase (PAL) gene 55.60 phenylpropanoid biosynthetic pathway 59-61 phloem transport under salinity 150-7 adding tracer to mature shoot tissue 152 calcium recirculation in the shoot 155-6 net fluxes and contents of xylem and phloem 1 5 2 4 pulsed labelling of roots 151-2 remobilization of nutrients from ageing shoot tissues 1 5 1 4 xyledphloem transfer 154-5 phosphate in soil 4, 13-14, 25 phospholipase C activity, inhibition of 5-6 phosphorus deficiency 25 extractable, in soil 4 salinization and transport of 13940, 158-60 short-term recirculation of 154-5

206

SUBJECT INDEX

phosphorus (cont.) transport in growing shoot tissues 144 uptake from soil 22, 25 phytochrome signal transduction 55, 56, 61-3 Phytophthora megasperma 60 Pinus muricuta 20, 21 Pisum 101 Pisum sativum 126 Poaceae 123, 136, 137, 149-50 polyphenols, effect on nitrification 20 potassium salinization, transport and 157 shoot accumulation 129 in soil 4 translocation to the shoot 129-30 transport in growing shoot tissues 144 transport in young tissue 133-7 potassium ion channels 5 primary response to salinization 115 Pseudomonus syringae pv. Glycinea 65

R Rhizobiuni 20 Ricinus communis 155, 159, 161 root signal hypothesis 118-1 19

S Saccharomyces cerevisiae 78 salicylic acid (SA) 55, 61 salinity in situ elemental and isotopic analysis 174-5 model systems 173-4 nutrient transport to growing shoot tissue 132-46 phloem transport and 150-7 reassessment of current status 171-3 shoot growth inhibition 115-21 shoot meristems 146-50 salinity tolerance 76-7 salinity toxicity 76-7 salinization cell division in leaves 125-6 cell extension 123 in dicots and monocots 121-6 nutrient transport disruptions 126-32 primordium formation and leaf emergence 123-5 timing of growth inhibition 121-3

whole shoot nutrient accumulation 128-32 salt-sensitive genotypeskell lines 103 salt tolerance 103, 117-18, 171-4 Scabiosa columbaria 5 Schizosaccharomyces pombe 78 secondary ion mass spectrometry (SIMS) 163, 167, 168-70 selectivity hypothesis 119 Shaker class of ion channel 84 short-term recirculation of nutrients 151-4 signal transduction networks ‘appropriate’ response 56-7 gene expression control by metabolites 57-9 gene expression responses and 54-5 interactions within 57-67 mechanisms in interactions between 67-9 negative regulation 57-63 networks vs pathways 55-6 phytochrome signal transduction pathways 61-3 plant defence responses 59-61 synergism 63-7 sodium carrier-mediated entry 80-2 channel-mediated entry 82-9 1 control levels of ions 127 co-residency of different channel types 89-9 1 effect of different channel types on rate of uptake 95-6 electrochemical potential differences 78-80 exclusion and genotypic tolerance 120-1 exclusion, uptake and sequestration of 77 influx across plasma membrane 101-2 ion selectivity of ion channels 82-4 semiquantitative dissection of fluxes 91-5 shoot accumulation 120 transport in young tissue 133-7 transport in growing shoot tissues 144 sodium absorption ratios (SARs) 128 sodium : calcium ratios 127-8 sodium chloride inhibition of shoot growth by 116-19, 173-4 disturbed photosynthesis I17 nutritional effect on shoot growth 119-21 soil water stress, ozone uptake and 33

207

SUBJECT INDEX

soil(s) acidic 4 calcareous 4 extractable elements 2 4 Solunum ruberosurn 41 Sorghum 144, 145 Sorxhurn bicolor 124, 132, 137, 138. 139, 140, 141, 149. 164 Spergrrlaria 80 stimulus-response pathways 56-7 Suuedu marititnu 152 sulphur dioxide emissions 32, 41, 48 synergism 63-7

T Trifoliuin alexundrinirm 124 TrijXurn pratense 124, 130, 131 Triricurn I 1 7, 132 Triticurn uesfivum 8, 13 I , 137, 139, 161 Triricum X Lophopyrum 120

U ultraviolet signal transduction pathway 65-7

V Vallisneria 81 Veronica qficina1i.s 13 Viciufubu 45 Vigna mungo 45 Vigna rudiata 45 Vignu unguiculutu 45 voltage-independent channels (VICs) 88-9

X xylem/phloem transfer 1.545

Z Zea 144, 145 Zru mays 117, 120, 121, 127, 130, 132, 138, 139, 140, 141, 159, 161

This Page Intentionally Left Blank